Compositions and methods for the removal of biofilms

ABSTRACT

This disclosure provides isolated or recombinant polypeptides that are useful to vaccinate individuals suffering from chronic/recurrent biofilm disease or as a therapeutic for those with an existing infection. The individual&#39;s immune system will then naturally generate antibodies which prevent or clear these bacteria from the host by interfering with the construction and or maintenance of a functional protective biofilm. Alternatively, antibodies to the polypeptides can be administered to treat or prevent infection. Bacteria that cannot form functional biofilms are more readily cleared by the remainder of the host&#39;s immune system and/or traditional antibiotics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. § 120 of U.S.application Ser. No. 15/999,215, filed Aug. 16, 2018, now U.S. Pat. No.11,274,144 dated Mar. 15, 2022, which is a divisional under 35 U.S.C. §120 of U.S. application Ser. No. 15/078,987, filed Mar. 23, 2016, nowabandoned, which claims priority under 35 U.S.C. § 119(e) to U.S.Application No. 62/199,952, filed Jul. 31, 2015, and U.S. applicationSer. No. 15/078,987 is a continuation-in-part under 35 U.S.C. § 120 ofU.S. application Ser. No. 14/967,228, filed Dec. 11, 2015, nowabandoned, which in turn is a continuation under 35 U.S.C. § 120 ofInternational Application No. PCT/US2014/042201, filed Jun. 12, 2014,which claims priority under 35 U.S.C. § 119(e) to U.S. Application No.61/834,846, filed Jun. 13, 2013, the content of each of which is herebyincorporated by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Contract No.R01DC011818 awarded by the National Institute of Deafness andCommunication Disorders (NIDCD) at the National Institutes of Health.The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 15, 2021, isnamed 106887-3895_SL.txt and is 179,389 bytes in size.

FIELD

The present disclosure generally relates to the methods and compositionsto lessen and/or cure bacterial biofilms.

BACKGROUND

The DNABII family of proteins are naturally found outside of thebacterial cell and contribute to biofilm formation. At least one proteinfrom the DNABII family is found in all known eubacteria. While theseproteins elicit a strong innate immune response, host subjects fail tonaturally produce immunoprotective antibodies to family members as aresult of infection. The major problem with bacterial biofilms is theinability of the host immune system and/or antibiotics and otherantimicrobials to gain access to the bacteria protected within thebiofilm.

Biofilms are present in an industrial setting as well. For example,biofilms are implicated in a wide range of petroleum process problems,from the production field to the gas station storage tank. In the field,sulfate reducing biofilm bacteria produce hydrogen sulfide (soured oil).In the process pipelines, biofilm activity develops slimes which impedefilters and orifices. Biofilm and biofilm organisms also cause corrosionof pipeline and petroleum process equipment. These problems can bemanifested throughout an oil or gas production facility, to the pointwhere fouling and corrosive biofilm organisms have even been found onthe surfaces of final product storage tanks.

Biofilms are implicated in a wide range of water processes, bothdomestic and industrial. They can grow on the surface of processequipment and impede the performance of the equipment, such asdegradation of heat transfer or plugging of filters and membranes.Biofilms growing on a cooling tower fill can add enough weight to causecollapse of the fill. Biofilms cause corrosion of even highlyspecialized stainless steels. Biofilms in a water process can degradethe value of a final product such as biofilm contamination in a paperprocess or the attachment of even a single cell on a silicon chip.Biofilms growing in drinking water distribution systems can harborpotential pathogenic organisms, corrosive organisms or bacteria thatdegrade the aesthetic quality of the water. In the home, biofilms arefound in or on any surface that supports microbial growth, e.g., indrains, on food preparation surfaces, in toilets, and in swimming poolsand spas.

Thus, a need exists to break through the protective barrier of biofilmsto treat or kill the associated bacterial infections and clear them fromsurfaces and in water systems.

DESCRIPTION OF TABLES

Tables 1-5 are the results of an in vitro bioassay of the reversal ofthe biofilm in the indicated organism.

Table 6 is the scoring scheme of the relative amount of biomass withinthe middle ear in the Chinchilla model of otitis media (OM).

Table 7 is the results of an in vitro bioassay of the reversal of thebiofilm upon treatment with DNase.

Table 8 is a non-limited summary of DNA binding proteins produced bygram (+) and gram (−) bacteria that can be used in the methods providedherein.

Table 9 is a listing of α, β, and C-terminal portions of DNABII proteinsfrom the indicated organism.

Table 10 is a listing of non-limiting exemplary hybridoma cell linesthat produce non-limiting exemplary monoclonal antibodies.

SUMMARY

Within bacterial cells, the DNABII proteins are DNA binding proteinsthat necessarily bend DNA substrates upon binding. Similarly, DNA thatis already in a bent conformation is an exemplary substrate as theenergy required for bending is rendered unnecessary.

The DNABII family is a member of a class of proteins referred to asnucleoid associated proteins (NAPs), bacterial proteins that, in part,shape the intracellular bacterial nucleoid (Browning et al. (2010) Curr.Opin. Microbiol. 13:773-780). In addition, this family is ubiquitous,expressed by virtually all eubacteria. All characterized family membersto date function as either a homodimer or heterodimer of subunits. Thefamily is divided into two types, HU (histone-like protein) and IHF(integration host factor) with B. cenocepacia capable of expressing both(strain J2315 genes: BCAL3530, hupA; BCAL1585, hupB; BCAL1487, ihfA andBCAL2949, ihfb). The primary distinction between these family members isthat HU binds DNA in a sequence independent manner, while IHF binds aconsensus sequence (WATCAANNNNTTR (SEQ ID NO: 36) where W is A or T andR is a purine) conserved across genera (Swinger et al. (2004) Curr.Opin. Struct. Biol. 14:28-35)]. All DNABII proteins bind to and bend DNAconsiderably e.g. E. coli IHF can bend DNA into a virtual U-turn (Riceet al. (1996) Cell 87: 1295-1306). In addition, all family members havea preference for pre-bent or curved DNA structures e.g. Hollidayjunctions, a cruciform-like structure central to DNA recombination. Infact, DNABII proteins function as accessory factors facilitating allintracellular DNA functions, including gene expression, recombination,repair and replication (Swinger et al. (2004) Curr. Opin. Struct. Biol.14:28-35).

The DNABII family of proteins is found outside of bacterial cells in thebiofilm state. Applicants have shown that these proteins are in factbound to the extracellular DNA at critical branched junctions. In oneaspect, Applicants have shown that by immunizing the host withpolypeptides and proteins that produce specific antibodies as well asadministrating antibodies and other interfering agents, the DNA-basedlattice is sufficiently altered to now permit the host immune system toclear the biofilm.

Applicants also have demonstrated the removal of pre-formed non-typeableHaemophilus influenzae biofilms in the middle ear of the chinchilla hostby various modes of immunization with a DNABII family member (E. coliintegration host factor, IHF). This chinchilla middle ear biofilm animalsystem has been well documented as an excellent model for human otitismedia (or middle ear infections).

The method for using this technology is straightforward. In oneembodiment, the polypeptides disclosed herein are used to vaccinateindividuals as a prophylactic to chronic/recurrent biofilm disease or asa therapeutic for those with an existing infection. The individual'simmune system will then clear the organism by use of the host's innateor acquired immune response, e.g., by binding of antibody andcomplement, phagocytosis, destruction by binding of effectors of innateimmunity etc. Alternatively, antibodies as well as fragments,derivatives, variants and interfering polynucleotides that bind to thepolypeptides or microbial DNA can be administered to treat or preventinfection. Bacteria that cannot form functional biofilms are morereadily cleared by the remainder of the host's immune system. In thisway, antibiotic and drug resistance of the bacteria can be reversed byallowing the therapeutic agents to reach the bacteria.

Thus, in one aspect a method for inhibiting, competing or titrating thebinding of a DNABII polypeptide or protein to a microbial DNA isprovided, the method comprising, or alternatively consisting essentiallyof, or yet further consisting of contacting the DNABII polypeptide orprotein or the microbial DNA with an interfering agent, therebyinhibiting, competing or titrating the binding of the DNABII protein orpolypeptide to the microbial DNA. The contacting can be performed invitro or in vivo.

In another aspect, provided is a method for inhibiting, preventing orbreaking down a microbial biofilm, comprising, or alternativelyconsisting essentially of, or yet further consisting of contacting thebiofilm with an interfering agent, thereby inhibiting, preventing orbreaking down the microbial biofilm. The contacting can be performed invitro or in vivo and therefore can break down, prevent, or inhibit abiofilm on surface or in an industrial or therapeutic setting.

In a further aspect, provided is a method of inhibiting, preventing orbreaking down a biofilm in a subject, comprising, or alternativelyconsisting essentially of, or yet further consisting of administering tothe subject an effective amount of an interfering agent, therebyinhibiting, preventing or breaking down the microbial biofilm.

In a yet further aspect, a method for inhibiting, preventing or treatinga microbial infection that produces a biofilm in a subject is provided.The method comprises, or alternatively consists essentially of or yetfurther consists of administering to the subject an effective amount ofan interfering agent, thereby inhibiting, preventing or treating themicrobial infection that produces the biofilm in the subject.

In one aspect, provided herein is an isolated antibody, an antigenbinding fragment, or an interfering agent that specifically recognizesor binds an isolated or recombinant DNABII polypeptide or a fragment oran equivalent each thereof. In one aspect, the isolated antibody,antigen binding fragment, or an interfering agent has affinity for atleast one DNABII protein that exceeds the affinity of the DNABII proteinfor components of a biofilm that includes the DNABII protein. In anotheraspect, the isolated antibody, antigen binding fragment, or aninterfering agent is selected from the group of: a monoclonal antibody,a bispecific antibody, a chimeric antibody, an antigen binding fragment,e.g., an Fv antibody, or a complete antibody that comprises constantregions heterologous to variable regions. In a further aspect, theantigen binding fragment comprises an isolated polypeptide or apolynucleotide. In one aspect, the biofilm component comprises branchedDNA. In another aspect the DNABII protein of the biofilm is selectedfrom the group of an IHF or a subunit thereof, HU protein, DPS, Hfq,CbpA or CbpB or a fragment or subunit thereof, e.g., a tip fragment ofthe protein. In a further aspect, the antibody, interfering agent orantigen binding fragment binds an epitope on the DNABII protein that isconserved across bacterial species. In another aspect, the antibody,antigen binding fragment or interfering agent dissolves a biofilmderived from at least two bacterial species, including both Grampositive and Gram negative species. In one aspect, the DNABII protein isStaphylococcus aureus HU or a fragment thereof; and optionally whereinthe fragment of Staphylococcus aureus HU comprises a tip fragment or abeta hairpin fragment or a biological equivalent thereof. In anotheraspect the antibody, antigen binding fragment or interfering agentdissolves a biofilm that is produced by S. aureus, P. aeruginosa and K.pneumonia. In another aspect, the affinity of the isolated antibody, anantigen binding fragment, or an interfering agent for the DNABII proteinis at least as strong as 100 pM or optionally 40 pM. In a furtheraspect, the antibody, the antigen binding fragment, or an interferingagent, produces an immune response that is immunodominant andimmunoprotective. In a further aspect, the antibody, the antigen bindingfragment, or the interfering agent specifically binds the tip fragmentof a DNABII polypeptide, e.g., the tip fragment of IHF or HUpolypeptide. In another aspect, the antibody, the antigen bindingfragment, or the interfering agent is a protein or a nucleic acid, theseare prepared for use in a method to a dissolve, interfere, prevent,treat or titrate a biofilm in a subject or to confer passively on asubject a capability to dissolve biofilms.

In a further aspect, a method is provided to prevent formation of or todisperse a biofilm associated with an industrial process by treating asurface or industrial environment (e.g., within a pipe) susceptible toor containing a biofilm with the antibody, antigen binding fragment, oran interfering agent as disclosed herein.

In another aspect, also provided is a polynucleotide encoding theantibody, antigen binding fragment, or the interfering agent asdescribed herein, wherein in one aspect, the polynucleotide is operablylinked to a regulatory element, that optionally is contained within anexpression vector or a host cell. When the polynucleotide encodes theantibody, antigen binding fragment, or the interfering agent, thepolynucleotide can be used to recombinantly produce the antibody,antigen binding fragment or interfering agent by culturing the cellsunder conditions to produce the antibody, antigen binding fragment orinterfering agent and isolating or purifying the product from the cellor cell culture. In one aspect the host cell is a mammalian cell.

Also provided herein is a method to identify a binding moiety, e.g., anantibody, antigen binding fragment or an interfering agent, that hasaffinity with at least one DNABII protein greater than the affinity of abiofilm component for the DNABII protein, by contacting a candidatebinding moiety with a biofilm component and with the at least one DNABIIprotein, and determining the ratio of the DNABII protein bound to saidbinding moiety as compared to DNABII bound with the biofilm component,whereby a ratio greater than one identifies a binding moiety that hasaffinity with respect to at least one DNABII protein greater than theaffinity of a biofilm component for the DNABII protein.

In another aspect, also provided is a method to identify an agent, e.g.,an antibody, an antigen binding agent or an interfering agent, thatreverses drug resistance in multiple species of bacteria by evaluatingan agent for activity in disrupting biofilms produced by multiplespecies, wherein an agent which disrupts said biofilms is identified asan agent that reverses drug resistance, and optionally whereinevaluating an agent for binding to DNABII proteins characteristic of amultiplicity of microbial species and wherein an agent that binds amultiplicity of said proteins is identified as an agent that reversesdrug resistance. In one aspect, the agent is effective against biofilmsderived from at least two bacterial species, including gram positive andgram negative species, non-limiting examples of such include S. aureus,P. aeruginosa, and K. pneumoniae.

Also provided herein is an isolated polypeptide comprising the aminoacid sequence of the Haemophilus influenzae IHFa or a fragment orequivalent thereof; and optionally wherein the polypeptide comprises:the tip portion of the DNABII polypeptide; the A5 fragment of IHFachain; or a polypeptide consisting of an amino acid sequence selectedfrom the group consisting of an amino acid sequence corresponding topositions 10-25 of the Haemophilus influenzae IHFa chain; an amino acidsequence corresponding to positions 56-78 of the Haemophilus influenzaeIHFa; or an amino acid sequence corresponding to positions 86-96 of theHaemophilus influenzae IHFa; or a polypeptide comprises the amino acidsequence selected from the group consisting of TFRPGQKLKSRVENASPKDE (SEQID NO: 252), MATITKLDIIEYLSDKYHLS (SEQ ID NO: 348),KYIILSKQDTKNVVENFLEEI (SEQ ID NO: 349), FLEEIRLSLESGQDVKLSGF (SEQ ID NO:350), KLSGFGNFELRDKSSRPGRN (SEQ ID NO: 351), RPGRNPKTGDVVPVSARRVV (SEQID NO: 352), ARRVVTFKPGQKLRARVEKTK (SEQ ID NO: 353), or a biologicalequivalent each thereof; and optionally wherein the isolated polypeptidefurther comprises a heterologous amino acid sequence or is coupled to aheterologous non-peptide domain.

In a further aspect, also provided is a non-physiological surface coatedwith the antibody, an antigen binding fragment, or an interfering agentas described herein. In one aspect the surface is in an industrialsetting. In a further aspect, the antibody, antigen binding fragment,further comprises, consists essentially of, or yet further consists of acarrier, such as a pharmaceutically acceptable carrier. In a furtheraspect, the composition further comprises, consisting essentially of, oryet further consists of an additional agent such as an antibiotic.

In a further aspect, also provided is a method to obtain antibodiesimmunoreactive with a DNABII protein, e.g., an IHF protein or togenerate B cells that secrete antibodies immunoreactive with the DNABIIprotein, e.g., an IHF protein, by administering the polypeptidesidentified herein to a subject and then recovering antibodies from thesubject; or recovering B cells that produce the antibodies from thesubject. In a further aspect, the method further comprises screening theB cells for secretion of an antibody with high affinity for a DNABII,e.g., IHF protein, thus identifying B cells that secrete antibodiesimmunoreactive with DNABII, e.g., IHF protein; and optionally isolatingDNA or mRNA encoding the antibodies from the cells.

Also provided is a method to treat or prevent a condition in a subjector detect the formation of a biofilm in the subject characterized by theformation of a biofilm, by treating the subject with the antibody, theantigen binding fragment or interfering agent as described herein. Inone aspect, when the biofilm is to be detected, the method furthercomprises observing complexation of the antibody, the antigen bindingfragment, or the interfering agent with any biofilm present. In oneaspect, the antibody, the antigen binding fragment, or the interferingagent dissolves biofilm derived from at least three bacterial species,wherein the three bacteria species comprises a gram negative or a grampositive bacteria. Non-limiting examples of such include S. aureus, P.aeruginosa, and K. pneumoniae. Non-limiting examples of conditionsinclude chronic non-healing wounds, including venous ulcers and diabeticfoot ulcers, ear infections, sinus infections, urinary tract infections,pulmonary infections, cystic fibrosis, chronic obstructive pulmonarydisease, catheter-associated infections, infections associated withimplanted prostheses, and periodontal disease.

In a yet further aspect, a method is provided to identify an immunogenuseful to elicit the formation of antibodies for treating, preventing,inhibiting or titrating a biofilm, by screening a library of candidateimmunogens for high affinity binding to an antibody, antigen bindingfragment or interfering agent as described herein, where a candidatemember of the library that binds with high affinity to the antibody,antigen binding fragment or interfering agent, is identified as animmunogen. In a further aspect, an antibody, antigen binding fragmentand interfering agent can be prepared by administering to a subject theimmunogen.

Yet further provided is a pharmaceutical composition for treating abiofilm in a subject or a condition characterized by formation ofbiofilms which comprises as active ingredient the antibody, an antigenbinding fragment, or an interfering agent as disclosed herein, in anamount effective to prevent or inhibit, disperse or dissolve a biofilmcharacteristic of the condition. In one aspect, the composition furtherincludes a pharmaceutically acceptable excipient, and optionally atleast one antibiotic.

Yet further provided is a method to prepare an interfering nucleic acidby preparing a nucleic acid consisting of 10-20 nucleotides thatspecifically binds a specific binding partner to antibody, an antigenbinding fragment, or an interfering agent as described herein. In afurther aspect, the specific binding partner is an epitope of a DNABIIprotein; and optionally wherein the epitope is conserved across at leastthree bacterial species. In a further aspect, this disclosure furtherprovides the interfering nucleic acid prepared by this method.

In a further aspect, also provided is a method to identify an antibody,antigen binding fragment or an interfering agent, e.g., a binding moietythat has affinity with respect to at least one DNABII protein greaterthan the affinity of a biofilm component for the DNABII protein, bycontacting a candidate antibody, antigen binding fragment, aninterfering agent, or binding moiety with a biofilm component and withthe at least one DNABII protein, and determining the ratio of saidDNABII protein bound to the antibody, antigen binding fragment, aninterfering agent or binding moiety as compared to DNABII bound with thebiofilm component, whereby a ratio greater than one identifies theantibody, antigen binding fragment, the interfering agent or the bindingmoiety that has affinity with respect to at least one DNABII proteingreater than the affinity of a biofilm component for the DNABII protein.In one aspect, the antibody, the antigen binding fragment, theinterfering agent or the binding moiety binds the DNABII protein in lowacid environments or over a range of pH.

Yet further provided is a small molecule that an antibody, an antigenbinding fragment, or an interfering agent that mimics the epitope towhich the antibody, antigen binding fragment or interfering agent asdescribed herein binds. In one aspect the epitope is a Staphylococcusaureus DNABII or a fragment thereof or a biological equivalent thereof;and optionally, wherein the fragment of Staphylococcus aureus DNABIIcomprises a beta hairpin fragment or a biological equivalent thereof.Yet further provided is a method to obtain antisera effective todissolve biofilm, by immunizing a subject with the small molecule thatmimics the epitope to which an antibody, an antigen binding fragment, oran interfering agent as described herein binds, and recovering antiserumfrom the subject, and optionally isolating polyclonal antiserum ormonoclonal antibodies derived therefrom. In a further aspect, theantisera is used to treat biofilm-related conditions in a subject, byadministering to the subject this antiserum or monoclonal antibodies.

Also provided is a method to image a biofilm by treating a biofilm withan antibody, an antigen binding fragment, or an interfering agent, e.g.,a monoclonal antibody or antigen binding fragment thereof. In oneaspect, the antibody or fragment is conjugated to an observable label,and an image is obtained using the label.

For the methods as described herein, any agent that interferes orimpedes the binding of the microbial DNA to the DNABII protein orpolypeptide is intended within the scope of this disclosure.Non-limiting examples of interfering agents include:

(a) an isolated or recombinant DNABII or an integration host factor(IHF) polypeptide or a fragment or an equivalent of each thereof;

(b) an isolated or recombinant histone-like protein from E. coli, e.g.,E. coli strain U93 (HU) polypeptide or a fragment or an equivalent ofeach thereof;

(c) an isolated or recombinant protein or polypeptide identified inTable 8, FIG. 19 , FIG. 20 , Table 9 or a DNA binding peptide identifiedin FIGS. 6A-6B, or a fragment or an equivalent of each thereof, whereinin one aspect the fragment comprises, or consists essentially of, or yetconsists of, the polypeptides identified as A1 through A6 or B1 throughB6 as disclosed in FIG. 18 ; or comprising, or alternatively consistingessentially of, or yet further consisting of the “tip” portion of theDNABII protein or polypeptide, non-limiting examples of such includepolypeptides comprising, or alternatively consisting essentially of, oryet further consisting of KLSGFGNFELRDKSSRPGRN (also referred to hereinas hIFA4; (SEQ ID NO. 351)); RGFGSFSLHHRQPRLGRNPK (also referred to B4(SEQ ID NO. 345)); RPGRNPKTGDVVPVSARRVV (also referred to herein ashIFA5; (SEQ ID NO. 352)); ARRVVTFKPGQKLRARVEKTK (also referred to hereinas hIFA6; (SEQ ID NO. 353)), or an equivalent of each thereof, or anequivalent of each thereof or a polypeptide having at least 60%, oralternatively at least 65%, or alternatively at least 70%, oralternatively at least 75%, or alternatively 80%, or alternatively atleast 85%, or alternatively at least 90%, or alternatively at least 95%identity thereto or for polypeptide sequences, which is encoded by apolynucleotide or its complement that hybridizes under conditions ofhigh stringency to a polynucleotide encoding such polypeptide sequences.Conditions of high stringency are described herein and incorporatedherein by reference. Applicants have determined that the bolded andunderlined amino acids are heavily conserved and therefore in oneaspect, are not modified or altered in designing an equivalentpolypeptide. Additional examples of equivalent polypeptides include, forexample, IEYLSDKYHLSKQDTK (SEQ ID NO. 354), DKSSRPGRNPKTGDVVAASARR (SEQID NO.: 355), and KLRARVEKTK (SEQ ID NO. 17), described in U.S. Ser.Nos. 14/497,147 and 14/668,767 now abandoned). Equivalent polypeptidesalso include a polypeptide consisting of or comprising the above notedpolypeptides with the addition of up to 25, or alternatively 20, oralternatively 15, or alternatively up to 10, or alternatively up to 5random amino acids on either the amine or carboxy termini (or on both);(d) an isolated or recombinant polypeptide of SEQ ID NO: 1 through 353,or a fragment or an equivalent of each thereof;(e) an isolated or recombinant C-terminal polypeptide of SEQ ID NO: 6through 11, 28, 29, 42 through 100, Table 8 or those C-terminalpolypeptides identified in Table 9 or a fragment or an equivalent ofeach thereof;(f) a polypeptide or polynucleotide that competes with a DNABII proteinon binding to a microbial DNA, e.g., DNABII or an integration hostfactor IHF and/or HU polypeptide or protein;(g) a four-way junction polynucleotide resembling a Holliday junction, a3 way junction polynucleotide resembling a replication fork, apolynucleotide that has inherent flexibility or bent polynucleotide;(h) an isolated or recombinant polynucleotide encoding any one of (a)through (f) or an isolated or recombinant polynucleotide of SEQ ID NO:36 or an equivalent of each thereof, or a polynucleotide that hybridizesunder stringent conditions to the polynucleotide its equivalent or itscomplement;(i) an antibody or antigen binding fragment that specifically recognizesor binds any one of (a) through (g), or an equivalent or fragment ofeach antibody or antigen binding fragment thereof;(j) an isolated or recombinant polynucleotide encoding the antibody orantigen binding fragment of (i) or its complement;(k) a small molecule that competes with the binding of a DNABII proteinor polypeptide to a microbial DNA;(l) an antibody or antigen binding fragment that specifically recognizesor binds any one of an isolated or recombinant polypeptide of SEQ ID NO:342 through 353 or a fragment or an equivalent of each thereof; and/or(m) polypeptide that comprises, or alternatively consists essentiallyof, or yet further consists of polypeptides A1 through A6 or B1 throughB6 as disclosed in FIG. 18 ; or KLSGFGNFELRDKSSRPGRN (also referred toherein as hIFA4; (SEQ ID NO. 351)); RPGRNPKTGDVVPVSARRVV (also referredto herein as hIFA5; (SEQ ID NO. 352)); ARRVVTFKPGQKLRARVEKTK (alsoreferred to herein as hIFA6; (SEQ ID NO. 353)), or an equivalent of eachthereof or a polypeptide having at least 60%, or alternatively at least65%, or alternatively at least 70%, or alternatively at least 75%, oralternatively 80%, or alternatively at least 85%, or alternatively atleast 90%, or alternatively at least 95% identity thereto or forpolypeptide sequences, which is encoded by a polynucleotide or itscomplement that hybridizes under conditions of high stringency to apolynucleotide encoding such polypeptide sequences. Conditions of highstringency are described herein and incorporated herein by reference.Applicants have determined that the bolded and underlined amino acidsare heavily conserved and therefore in one aspect, are not modified oraltered in designing an equivalent polypeptide. Additional examples ofequivalent polypeptides include, for example IEYLSDKYHLSKQDTK (SEQ IDNO. 354), DKSSRPGRNPKTGDVVAASARR (SEQ ID NO.: 355), AND KLRARVEKTK (SEQID NO. 17) described in U.S. Ser. Nos. 14/497,147 and 14/668,767 nowabandoned), a polypeptide consisting of or comprising the above notedpolypeptides with the addition of up to 25, or alternatively 20, oralternatively 15, or alternatively up to 10, or alternatively up to 5random amino acids on either the amine or carboxy termini (or on both).

Also provided herein is a method for inducing an immune response in orconferring passive immunity in a subject in need thereof, comprising, oralternatively consisting essentially of or yet further consisting of,administering to the subject an effective amount of one or more agentsof the group:

(a) an isolated or recombinant DNABII or an integration host factor(IHF) polypeptide, or a fragment or an equivalent of each thereof;

(b) an isolated or recombinant histone-like protein from E. coli, e.g.,E. coli strain U93 (HU) polypeptide or a fragment or an equivalent ofeach thereof;

(c) an isolated or recombinant protein polypeptide identified in Table8, FIG. 19 , FIG. 20 , Table 9 or an DNA binding peptide identified inFIGS. 6A-6B, or a fragment or an equivalent of each thereof, apolypeptide A1 to A6, or B1 to B6 as shown in FIG. 18 , or a fragmentthat comprises, or consists essentially of, or yet consists of, the“tip” portion of the DNABII protein or polypeptide, non-limitingexamples of such include without limitation a polypeptide thatcomprises, or alternatively consists essentially of, or yet furtherconsists of KLSGFGNFELRDKSSRPGRN (also referred to herein as hIFA4; (SEQID NO. 351)); RGFGSFSLHHRQPRLGRNPK (also referred to B4 (SEQ ID NO.345)); RPGRNPKTGDVVPVSARRVV (also referred to herein as hIFA5; (SEQ IDNO. 352)); ARRVVTFKPGQKLRARVEKTK (also referred to herein as hIFA6; (SEQID NO. 353)), or an equivalent of each thereof. Additional examples ofequivalent polypeptides include, for example IEYLSDKYHLSKQDTK (SEQ IDNO. 354), DKSSRPGRNPKTGDVVAASARR (SEQ ID NO.: 355), AND KLRARVEKTK (SEQID NO. 17) described in U.S. Ser. Nos. 14/497,147 and 14/668,767 nowabandoned), a polypeptide consisting of or comprising the above notedpolypeptides with the addition of up to 25, or alternatively 20, oralternatively 15, or alternatively up to 10, or alternatively up to 5random or naturally occurring amino acids on either the amine or carboxytermini (or on both);(d) an isolated or recombinant polypeptide of SEQ ID NO: 1 through 353or a fragment or an equivalent thereof;(e) an isolated or recombinant C-terminal polypeptide of SEQ ID NO: 6through 11, 28, 29, 42 through 100, Table 8 or those C-terminalpolypeptides identified in Table 9 or a fragment or an equivalent ofeach thereof;(f) an isolated or recombinant polynucleotide encoding any one of (a)through (e) or an isolated or recombinant polynucleotide SEQ ID NO: 36or an equivalent of each thereof, or a polynucleotide that hybridizesunder stringent conditions to the polynucleotide, its equivalent or itscomplement;(g) an antibody or antigen binding fragment that specifically recognizesor binds any one of (a) through (e), or an equivalent or fragment ofeach thereof;(h) an isolated or recombinant polynucleotide encoding the antibody orantigen binding fragment of (g);(i) an antigen presenting cell pulsed with any one of (a) through (e);(j) an antigen presenting cell transfected with one or morepolynucleotides encoding any one of (a) through (e); and/or(k) an antibody or antigen binding fragment that specifically recognizesor binds any one of an isolated or recombinant polypeptide noted hereinas A1, A2, A3, A4, A5, A6, B1, B2, B3, B4, B5, B6 or an isolated orrecombinant polypeptide of SEQ ID NO: 342 through 353 or a fragment oran equivalent of each thereof.

Subjects in need of such immune response include those at risk of orsuffering from an infection that produces a microbial biofilm.

Also provided herein are compositions for use in the above methods,non-limiting examples of which are discussed below.

In one aspect, provided is an isolated or recombinant polypeptidecomprising, or alternatively consisting essentially of an amino acidsequence selected from A1, A2, A3, A4, A5, A6, B1, B2, B3, B4, B5, B6(see FIG. 18 ) or SEQ ID NO: 1 to 5 or 12 to 27, 30 to 35, 101-340, or341-353, or a DNA binding peptide identified in FIGS. 6A-6B, or a DNABIIfragment that comprises, or consists essentially of, or yet consists of,the “tip” portion of the DNABII protein or polypeptide, non-limitingexamples of such include without limitation a polypeptide thatcomprises, or alternatively consist essentially of, or yet furtherconsists of KLSGFGNFELRDKSSRPGRN (also referred to herein as hIFA4; (SEQID NO. 351)); RPGRNPKTGDVVPVSARRVV (also referred to herein as hIFA5;(SEQ ID NO. 352)); ARRVVTFKPGQKLRARVEKTK (also referred to herein ashIFA6; (SEQ ID NO. 353)); RGFGSFSLHHRQPRLGRNPK (also referred to B4 (SEQID NO. 345)); or an equivalent of each thereof. An example of equivalentpolypeptides include, for example IEYLSDKYHLSKQDTK (SEQ ID NO. 354),DKSSRPGRNPKTGDVVAASARR (SEQ ID NO.: 355), and KLRARVEKTK (SEQ ID NO. 17)described in U.S. Ser. Nos. 14/497,147 and 14/668,767 now abandoned),antibody sequences as provided herein, a polypeptide consisting of orcomprising the above noted polypeptides with the addition of up to 25,or alternatively 20, or alternatively 15, or alternatively up to 10, oralternatively up to 5 random or naturally occurring amino acids oneither the amine or carboxy termini (or on both), or an equivalent ofeach thereof, or an equivalent of each thereof or a polypeptide havingat least 60%, or alternatively at least 65%, or alternatively at least70%, or alternatively at least 75%, or alternatively 80%, oralternatively at least 85%, or alternatively at least 90%, oralternatively at least 95% identity thereto or for polypeptidesequences, which is encoded by a polynucleotide or its complement thathybridizes under conditions of high stringency to a polynucleotideencoding such polypeptide sequences. Conditions of high stringency aredescribed herein and incorporated herein by reference. Applicants havedetermined that the bolded and underlined amino acids are heavilyconserved and therefore in one aspect, are not modified or altered indesigning an equivalent polypeptide. Additional examples of equivalentpolypeptides include, for example IEYLSDKYHLSKQDTK (SEQ ID NO. 354),DKSSRPGRNPKTGDVVAASARR (SEQ ID NO.: 355), and KLRARVEKTK (SEQ ID NO. 17)described in U.S. Ser. Nos. 14/497,147 and 14/668,767 now abandoned).Equivalent polypeptides also include a polypeptide consisting of orcomprising the above noted polypeptides with the addition of up to 25,or alternatively 20, or alternatively 15, or alternatively up to 10, oralternatively up to 5 random amino acids on either the amine or carboxytermini (or on both).

In another aspect, provided is an isolated or recombinant polypeptidecomprising, or alternatively consisting essentially of, or yet furtherconsisting of SEQ ID NO: 1 or 2, with the proviso that the polypeptideis none of SEQ ID NO: 6 to 11, 28, 29, or 42 through 100.

In one aspect, provided is an isolated or recombinant polypeptidecomprising, or alternatively consisting essentially of, or yet furtherconsisting of SEQ ID NO: 3, 4 or 5, with the proviso that thepolypeptide is none of SEQ ID NO: 6 to 11, 28, 29, or 42 through 100.

In one aspect, provided is an isolated or recombinant polypeptidecomprising, or alternatively consisting essentially of, or yet furtherconsisting of, SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, 26, 30 or 32, withthe proviso that the polypeptide is none of SEQ ID NO: 6 to 11, 28, 29,or 42 through 100.

In one aspect, provided is an isolated or recombinant polypeptidecomprising, or alternatively consisting essentially of, or yet furtherconsisting of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 31 or 33, withthe proviso that the polypeptide is none of SEQ ID NO: 6 to 11, 28, 29,or 42 through 100.

In one aspect, provided is an isolated or recombinant polypeptidecomprising, or alternatively consisting essentially of, or yet furtherconsisting of SEQ ID NO: 337, 338, 339, or 340, with the proviso thatthe polypeptide is none of SEQ ID NO: 6 to 11, 28, 29, or 42 through100.

In one aspect, provided is an isolated or recombinant polypeptidecomprising, or alternatively consisting essentially of, or yet furtherconsisting of, SEQ ID NO: 12 and 13 or 14 and 15 or 16 and 17 or 18 and19 or 20 and 21 or 22 and 23 or 24 and 25, or 26 and 27 or 30 and 31 or32 and 33, with the proviso that the polypeptide is none of SEQ ID NO: 6to 11, 28, 29, or 42 through 100.

In one aspect, provided is an isolated or recombinant polypeptidecomprising, or alternatively consisting essentially of, or yet furtherconsisting of, one or more of the polypeptides noted above, nonelimiting examples of such include polypeptides containing the “tip”portion, the “tail portion” or the C-terminal region containing at least10, or alternatively at least 15, or alternatively at least 20, oralternatively at least 25, or alternatively at least 30, C-terminalamino acids of a polypeptide of the group of a DNABII polypeptide, anIHF polypeptide, a DPS polypeptide, an Hfq polypeptide, a CbpApolypeptide, a CbpB polypeptide, an HU polypeptide, SEQ ID NO: 6 through11, 28, 29 or those identified in Table 8, Table 9 or a fragment or anequivalent of each thereof.

Also provided are the polynucleotides encoding the polypeptides,recombinant expression systems and host cells comprising thepolynucleotides and use of same for the recombinant expression of thepolypeptides as well as the recombinant polypeptides encoding by thesystem and host cells. In one aspect, the host cell is a mammalian cell.The isolated polynucleotides can be operatively linked to regulatoryelements necessary for the expression and/or replication of thepolynucleotide. The polynucleotide can be contained within a vector. Inone aspect, the polynucleotides further comprise an artificial ornon-naturally occurring label (e.g., excluding naturally fluorescentpolynucleotides) bound to the polynucleotide for use as probes for usein detection of biofilms and monitoring of biofilm treatment.

In one aspect, provided is an isolated or recombinant polypeptide of thegroup of:

a polypeptide comprising SEQ ID NO: 12 and 13;

a polypeptide comprising SEQ ID NO: 14 and 15;

a polypeptide comprising SEQ ID NO: 16 and 17;

a polypeptide comprising SEQ ID NO: 18 and 19;

a polypeptide comprising SEQ ID NO: 20 and 21;

a polypeptide comprising SEQ ID NO: 23 and 24;

a polypeptide comprising SEQ ID NO: 25 and 26;

a polypeptide comprising SEQ ID NO: 30 and 31;

a polypeptide comprising SEQ ID NO: 32 and 33;

a polypeptide comprising SEQ ID NO: 34 and 35;

a polypeptide comprising SEQ ID NO: 337 and 338; or

a polypeptide comprising SEQ ID NO: 339 and 340;

a polypeptide consisting essentially of any one or more of SEQ ID NO:342 to 453;

with the proviso that the polypeptide is none of wild-type of any one ofIHF alpha, IHF beta or SEQ ID NO: 6 to 11, 28, 29, or 42 through 100.

In one aspect, provided is an isolated or recombinant polypeptide of thegroup of:

a polypeptide consisting essentially of SEQ ID NO: 12 and 13;

a polypeptide consisting essentially of SEQ ID NO: 14 and 15;

a polypeptide consisting essentially of SEQ ID NO: 16 and 17;

a polypeptide consisting essentially of SEQ ID NO: 18 and 19;

a polypeptide consisting essentially of SEQ ID NO: 20 and 21;

a polypeptide consisting essentially of SEQ ID NO: 23 and 24;

a polypeptide consisting essentially of SEQ ID NO: 25 and 26;

a polypeptide consisting essentially of SEQ ID NO: 30 and 31;

a polypeptide consisting essentially of SEQ ID NO: 32 and 33;

a polypeptide consisting essentially of SEQ ID NO: 34 and 35;

a polypeptide consisting essentially of SEQ ID NO: 337 and 338; or

a polypeptide consisting essentially of SEQ ID NO: 339 and 340;

a polypeptide consisting essentially of any one or more of SEQ ID NO:342 to 453;

with the proviso that the polypeptide is none of wild-type of any one ofIHF alpha, IHF beta or SEQ ID NO: 6 to 11, 28, 29, or 42 through 100.

Also provided are isolated or recombinant polypeptides comprising, oralternatively consisting essentially of or yet further consisting of,two or more, or three or more or four or more, or multiples of theabove-identified isolated polypeptides, including fragments andequivalents thereof. Examples of such include isolated polypeptidescomprising SEQ ID NO: 1 through 4 and/or 12 through 29, and/or 30through 33, and/or 30 through 35 e.g., SEQ ID NO: 1 and 2, oralternatively 1 and 3 or alternatively 1 and 4, or alternatively 2 and3, or alternatively SEQ ID NO: 1, 2 and 3 or alternatively, 2, 3 and 4,or alternatively 1, 3 and 4 or equivalent polypeptides, examples ofwhich are shown in Table 9. The polypeptides can be in any orientation,e.g., SEQ ID NO: 1, 2, and 3 or SEQ ID NO: 3, 2 and 1 or alternativelySEQ ID NO: 2, 1 and 3, or alternatively, 3, 1 and 2, or alternatively 11and 12, or alternatively 1 and 12, or alternatively 2 and 12, oralternatively, 1 and 12, or alternatively 2 and 13, or alternatively 12,16 and 1, or alternatively 1, 16 and 12.

In another aspect, this disclosure provides an isolated or recombinantpolypeptide comprising SEQ ID NO: 1 or 2 and 3 or 4 or a polypeptide orrecombinant polypeptide comprising, or alternatively consistingessentially of, or yet further consisting of an amino acid correspondingto fragments of a DNABII protein such as the tail fragment or the tipfragment, non-limiting examples of such include without limitation apolypeptide that comprises one or more of the sequences identified as A1through A6; or identified as B1 through B6 (see FIG. 18 );MATITKLDIIEYLSDKYHLS (also referred to herein as hIFA1; (SEQ ID NO.348)); KYHLSKQDTKNVVENFLEEI (also referred to herein as hIFA2; (SEQ IDNO. 349)); FLEEIRLSLESGQDVKLSGF (also referred to herein as hIFA3; (SEQID NO. 350)); KLSGFGNFELRDKSSRPGRN (also referred to herein as hIFA4;(SEQ ID NO. 351)); RPGRNPKTGDVVPVSARRVV (also referred to herein ashIFA5; (SEQ ID NO. 352)); ARRVVTFKPGQKLRARVEKTK (also referred to hereinas hIFA6; (SEQ ID NO. 353)); RGFGSFSLHHRQPRLGRNPK (also referred to B4(SEQ ID NO. 345)); Applicants have determined that the bolded andunderlined amino acids are heavily conserved and therefore in oneaspect, are not modified or altered in designing an equivalentpolypeptide, or an equivalent thereof, (examples of equivalentpolypeptides include, for example IEYLSDKYHLSKQDTK (SEQ ID NO. 354),DKSSRPGRNPKTGDVVAASARR (SEQ ID NO.: 355), and KLRARVEKTK (SEQ ID NO. 17)described in U.S. Ser. Nos. 14/497,147 and 14/668,767 now abandoned), apolypeptide consisting of or comprising the above noted polypeptideswith the addition of up to 25, or alternatively 20, or alternatively 15,or alternatively up to 10, or alternatively up to 5 random or naturallyoccurring amino acids on either the amine or carboxy termini (or onboth). the tail fragment, the β-3 and/or α-3 fragments of a DPSpolypeptide, an Hfq polypeptide, a CbpA polypeptide, a CbpB polypeptide,an HU polypeptide, a Haemophilus influenzae IHFα or IHFβ, non-limitingexamples of which include SEQ ID NO: 12 through 27, or a fragment or anequivalent of each of the polypeptides, examples of which are shown inTable 9. In one aspect, isolated wildtype polypeptides are specificallyexcluded, e.g., that the polypeptide is none of SEQ ID NO: 6 through 11or a wildtype sequence identified in Table 8. In this embodiment, SEQ IDNO: 1 or 2 or a polypeptide comprising, or alternatively consistingessentially of, or yet further consisting of an amino acid correspondingto the β-3 and/or α-3 fragments of an IHFα or IHFβ microorganism,non-limiting examples of which include SEQ ID NO: 12 through 27 and 30through 33 or an equivalent of each thereof is located upstream or aminoterminus from SEQ ID NO: 3 or 4 or a fragment or an equivalent thereof.In another aspect, the isolated polypeptide comprises SEQ ID NO: 3 or 4or a polypeptide comprising, or alternatively consisting essentially of,or yet further consisting of an amino acid corresponding to the β-3and/or α-3 fragments of an IHFα or IHFβ microorganism, non-limitingexamples of which include SEQ ID NO: 12 through 27, or an equivalentthereof located upstream or amino terminus to SEQ ID NO: 1 or 2 or anequivalent thereof.

In any of the above embodiments, a peptide linker can be added to thetip fragment (non-limiting examples of such include without limitation apolynucleotide that comprises one or more of the sequences:RPGRNPKTGDVVPVSARRVV (also referred to herein as hIFA5; (SEQ ID NO.352)); and RGFGSFSLHHRQPRLGRNPK (also referred to B4 (SEQ ID NO. 345));or an equivalent of each thereof, the tail fragment, the N-terminus orthe C-terminus of the polypeptide, fragment or equivalent thereof. Inone aspect, the linker joins the polypeptides disclosed herein, e.g.,SEQ ID NO: 1 to 4, 28, 29, 34, or 35 or 30 to 33, 34, or 35 or apolypeptide comprising, or alternatively consisting essentially of, oryet further consisting of an amino acid corresponding to the β-3 and/orα-3 fragments of a Haemophilus influenzae IHFα or IHFβ protein(non-limiting examples of full length Haemophilus influenzae IHFα andIHFβ are disclosed as SEQ ID NO: 6 and SEQ ID NO: 341, respectively);non-limiting examples of the polypeptides disclosed herein above includeSEQ ID NO: 12 through 27 or an equivalent of each thereof. The apolypeptides can further consists of or comprise the above notedpolypeptides with the addition of up to 25, or alternatively 20, oralternatively 15, or alternatively up to 10, or alternatively up to 5random or naturally occurring amino acids on either the amine or carboxytermini (or on both).

A “linker” or “peptide linker” refers to a peptide sequence linked toeither the N-terminus or the C-terminus of a polypeptide sequence. Inone aspect, the linker is from about 1 to about 20 amino acid residueslong or alternatively 2 to about 10, about 3 to about 5 amino acidresidues long. An example of a peptide linker is Gly-Pro-Ser-Leu-Lys-Leu(SEQ ID NO: 37).

Further provided is a fragment or an equivalent of the isolated orrecombinant polypeptide of any one of polypeptides identified above aswell as an isolated or recombinant polypeptide comprising, oralternatively consisting essentially of, or yet further consisting of,two or more of the isolated or recombinant polypeptides identifiedabove.

Yet further provided is a polynucleotide that interferes with thebinding between the microbial DNA and a polypeptide or fragment orequivalent thereof, e.g., SEQ ID 36, or a four-way junctionpolynucleotide resembling a Holliday junction, a 3 way junctionpolynucleotide resembling a replication fork, a polynucleotide that hasinherent flexibility or bent polynucleotide; an isolated or recombinantpolynucleotide encoding a polypeptide described above or an antibody orfragment thereof, which can be operatively linked to regulatory elementsnecessary for the expression and/or replication of the polynucleotide.The polynucleotide can be contained within a vector.

Also provided is an isolated host cell comprising, or alternativelyconsisting essentially of, or yet further consisting of an isolated orrecombinant polypeptide described above, a four-way junctionpolynucleotide resembling a Holliday junction, a 3 way junctionpolynucleotide resembling a replication fork, a polynucleotide that hasinherent flexibility or bent polynucleotide; an isolated or recombinantpolynucleotide as described above, or a vector as described above.

In one aspect the cell is an isolated antigen presenting cell comprisingthe isolated or recombinant polypeptide. In a further aspect, thepolypeptide is present on the surface of the cell, such as a dendriticcell. In a further aspect, the antigen presenting cell is transfectedwith one or more polynucleotides encoding the polypeptide.

Yet further provided is an antibody or antigen binding fragment thatspecifically recognizes and binds the isolated or recombinantpolypeptide as describe above, including a fragment or an equivalent ofthe polypeptide. Non-limiting examples of antibodies include apolyclonal antibody, a monoclonal antibody, a humanized antibody, ahuman antibody, an antibody derivative, a veneered antibody, a diabody,a chimeric antibody, an antibody derivative, a recombinant humanantibody, or an antibody fragment. In a particular aspect, the antibodyis a monoclonal antibody. Yet further provided is a hybridoma cell linethat produces the monoclonal antibody.

In certain aspects, the disclosure relates to an antibody or antigenbinding fragment that specifically recognizes or binds an isolated orrecombinant polypeptide comprising, consisting essentially of, orconsisting of one or more amino acid sequence selected from A1 to A6 orB1 to B6 (See FIG. 18 ); SEQ ID NO: 342-353, MATITKLDIIEYLSDKYHLS (alsoreferred to herein as hIFA1; (SEQ ID NO. 348)); KYHLSKQDTKNVVENFLEEI(also referred to herein as hIFA2; (SEQ ID NO. 349));FLEEIRLSLESGQDVKLSGF (also referred to herein as hIFA3; (SEQ ID NO.350)); KLSGFGNFELRDKSSRPGRN (also referred to herein as hIFA4; (SEQ IDNO. 351)); RPGRNPKTGDVVPVSARRVV (also referred to herein as hIFA5; (SEQID NO. 352)); ARRVVTFKPGQKLRARVEKTK (also referred to herein as hIFA6;(SEQ ID NO. 353)); RGFGSFSLHHRQPRLGRNPK (also referred to herein as B4(SEQ ID NO. 345)); or the tip or the tail portion of the DNABII proteinor polypeptide, a polypeptide that comprises one or more of thesequences MATITKLDIIEYLSDKYHLS (also referred to herein as hIFA1; (SEQID NO. 348)); KYHLSKQDTKNVVENFLEEI (also referred to herein as hIFA2;(SEQ ID NO. 349)); or FLEEIRLSLESGQDVKLSGF (also referred to herein ashIFA3; (SEQ ID NO. 350)). The polypeptides may consist of or comprisethe above noted polypeptides with the addition of up to 25, oralternatively 20, or alternatively 15, or alternatively up to 10, oralternatively up to 5 random or naturally occurring amino acids oneither the amine or carboxy termini (or on both). In some embodiments,such antibodies or antigen binding fragments are administered alone orin combination with each other, or an agent other than the antibody, oryet a further pharmaceutically effective agent, alone or in combinationwith a pharmaceutically acceptable carrier.

This disclosure also provides isolated or recombinant polynucleotidesencoding one or more of the above-identified isolated or recombinantpolypeptides or antibodies or a fragment thereof. Vectors comprising theisolated polynucleotides are further provided. In one aspect where morethan one isolated polypeptide disclosed herein, the isolatedpolynucleotides can be contained within a polycistronic vector.

Isolated host cells comprising one or more of isolated or recombinantpolypeptides or isolated or recombinant polynucleotides or the vectors,described herein are further provided. In one aspect the isolated hostcell is a prokaryotic cell or eukaryotic cell such as antigen presentingcell, e.g., a dendritic cell.

The polynucleotides, polypeptides, antibodies, antigen binding fragment,vectors or host cells can father comprise a detectable label.

Compositions comprising a carrier and one or more of an isolated orrecombinant polypeptide disclosed herein, an isolated or recombinantpolynucleotide disclosed herein, a vector disclosed herein, an isolatedhost cell disclosed herein, or an antibody of the embodiments are alsoprovided. The carriers can be one or more of a solid support, a medicaldevice like a stent or dental implant, or a liquid such as apharmaceutically acceptable carrier. The compositions can furthercomprise an adjuvant, an antimicrobial or an antigenic peptide.

The compositions can further comprise additional biologically activeagents. A non-limiting example of such is a antimicrobial agent such asother vaccine components (i.e., antigenic proteins or peptides) such assurface antigens, e.g., an OMP P5, rsPilA, OMP 26, OMP P2, or Type IVPilin protein (see Jurcisek and Bakaletz (2007) J. Bacteriology189(10):3868-3875 and Murphy, T. F. et al. (2009) The PediatricInfectious Disease Journal 28:S121-S126) and antimicrobial agents.

This disclosure also provides a method for producing an antigenicpeptide by growing or culturing a host cell comprising an isolatedpolynucleotide encoding an antigenic peptide as described above underconditions that favor the expression of the polynucleotide. Thepolypeptide produced by this method can be isolating for further invitro or in vivo use.

Also provided are hybridoma cell lines that produce exemplary monoclonalantibodies for use in the methods disclosed herein. The hybridoma celllines that produce monoclonal antibodies that specifically recognize andbind Haemophilus influenzae IhfA fragment A5 (SEQ ID NO. 352)), IhfB4(RGFGSFSLHHRQPRLGRNPK (also referred to B4 (SEQ ID NO. 345)); weredeposited with American Type Culture Collection (ATCC) under AccessionNumbers (IhfA5 (Accession No. PTA-122334)) and (IhfB4 (Accession No.PTA-122336)), pursuant to the provisions of the Budapest Treaty on Jul.30, 2015. Further non-limiting exemplary antibodies include those thatspecifically recognize and bind Haemophilus influenzae IhfA fragment A3(SEQ ID NO. 350), IhfB fragment 132 (SEQ ID NO. 343) produced byhybridoma cell lines IhfA3 NTHI 9B10.F2.H3 and IhfB2 NTHI 7A4.E4.G4.

A kit is also provided for diagnostic or therapeutic use comprising acomposition as described above and instructions for use. A kit is alsoprovided to perform screens for new drugs and/or combination therapiesas provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a biofilm formed in the chinchilla middle ear by NTHI strain86-028NP and labeled for NTHI Tfp pilin protein (appears as white orlight gray speckles and small clusters in the background of this image),as well as with DAPI for labeling of the double stranded DNA (dsDNA)(appears as dark gray overlapping strands and bundles of material withintermittent clumps in the foreground of this image). This figure hasbeen reproduced from Jurcisek and Bakaletz (2007) J. Bacteriology189(10):3868-3875. FIG. 1B shows immunolabeling bronchoalveolar lavage(BAL) from the lung of a child with cystic fibrosis. The lung of a childwith cystic fibrosis was washed out via BAL and the particulate matterfrom the wash was frozen and affixed to slides for immunolabeling. Thefrozen particulate matter was immunolabeled with the anti-IHF antibody.The presence of DNABII positive foci in the biofilm of human cysticfibrosis patients exemplifies that the etiology of human cystic fibrosisincludes a biofilm with IHF at the vertices of the dsDNA. FIG. 1C showssecretions from human sinus collected at the time of sinus surgery andembedded in OCT freezing medium (Optimal Cutting Temperature medium,available commercially from Fisher Scientific Cat. No. 14-373-65). 10 μmfrozen sections were cut and labeled with anti-IHF (appearing as grayclusters). dsDNA within the sample was stained with a fluorescent stain,DAPI (4′,6-diamidino-2-phenylindole, available commercially fromInvitrogen). The presence of IHF positive foci in the biofilm ofsinusitis patients exemplifies that the etiology of human sinusitisincludes a biofilm with IHF at the vertices of the dsDNA.

FIG. 2 is an immunohistochemical labeling of double stranded DNA(appears as white strands in this image) within an NTHI biofilm formedin the middle ear of chinchilla. Positive labeling for IHF (as indicatedby arrows pointing to punctuate foci in the middle panel of this 3-panelimage) was observed at nearly 100% of vertices formed by dsDNA. Meandistance between vertices was approximately 6 μm, or approximately 18 kbbetween each vertex if one assumes 0.34 nm per base of DNA for B-formDNA. To the best of our knowledge, the only proteins that possessepitopes that cross-react with anti-IHF are HU and IHF. Therefore, basedon these observations, it appeared that not only were thereextracellular DNABII proteins within the NTHI biofilm matrix, but moreimportantly that these proteins appeared to be exclusively positioned oneDNA strands that resembled cruciform structures in conformation (seeFIG. 6B, bottom section), thus strongly suggesting their role inmediating the resulting bent conformation of the eDNA.

FIGS. 3A-3E shows that antibodies directed against IHF reversed anestablished NTHI formed in a chamber slide. FIG. 3A shows a biofilmtreated with nonspecific antibody. FIG. 3B shows a biofilm treated withnaïve rabbit serum. Note the robust NTHI biofilm with abundant towers(appear as white to light gray clustered areas) and water channels(black spaces). FIG. 3C is a biofilm treated with anti-IHF. Note theeradication of biofilm structure after treatment with an anti-IHF.Individual NTHI (appear as small punctuate white to light gray spots)and sparse, short towers (appear as denser white to light gray clusteredareas) remain. FIGS. 3D and 3E further depict that antibodies directedagainst IHF reverse an established NTHI biofilm. As shown in FIG. 3E,the biofilm showed a dramatic loss of 3-dimensional structure whencompared to a biofilm incubated with naive serum (FIG. 3D), Via COMSTATanalysis of multiple replicate assays, the measured parameters ofbiofilm height, biomass and biofilm thickness were all diminished by amean of greater than 80% upon incubation with anti-IHF.

FIGS. 4A and 4B are graphs showing treatment of an established biofilmformed by NTHI with anti-IHF results in more NTHI released into thesupernatant. Sixteen (16) hour NTHI biofilms grown in chamber slideswere sham treated with sterile medium (sBHI) or treated with naïverabbit serum or rabbit anti-IHF. Six hours later (FIG. 4A) or 10 hourslater (FIG. 4B), supernatants were collected and analyzed. Note thegreater number of NTHI in supernatant after treatment with anti-IHF.Incubation with anti-IHF resulted in a marked increase in planktonicbacteria available for culture from the medium within the chamber slidewithin approximately 6 hrs, and increasing notably at 10 hours ofincubation. These results suggested release of bacteria from the biofilmmatrix.

FIG. 5 shows the results of a transcutaneous immunization with IHFreduced an established biofilm in the middle ear. Note that bullae wereblindly ranked onto a 0 to 4+ scale of relative remaining biofilm mass.

FIG. 6A is a map indicating the amino acid residues of IHF that interactwith or bind to another IHF in an IHF-IHF dimer (indicated by trianglesat the upper level) or interact with or bind DNA (indicated by trianglesat the lower level). The peptide is divided by the short vertical barsinto regions containing 3 amino acids. FIG. 6B graphically depicts theinteraction of microbial DNA with an IHF.

FIG. 7 depicts the reduction of biofilms formed by S. aureus, N.gonorrhoeae and P. aeruginosa upon incubation with rabbit anti-IHFcompared to incubation with naïve rabbit serum.

FIGS. 8A-8F show the effect of incubation of biofilms formed in vitro byE. coli with either naïve serum or with anti-IHF serum. Representativeimages of biofilms are shown in FIGS. 8A-8F with height of individualbiofilms shown to the right of each image, whereas mean values in termsof percent reduction in biofilm height, biomass and mean thickness asmediated by incubation with anti-IHF are shown in the tables at the endof each row. FIGS. 8A & 8B—parental strain MG1655; FIGS. 8C &8D—HU-deficient hupA, hupB double mutant; FIGS. 8E & 8F—IHF-deficienthimD, himA double mutant. Note the ability of anti-IHF serum to reducebiomass induced by either the parental isolate or the HU-deficientmutant, but not the IHF-deficient strain, as expected.

FIG. 9A demonstrates that immunization with IHF via a transcutaneousdelivery route induced the formation of antibodies that significantlyreduced the biomass of an NTHI-induced biofilm resident within themiddle ears of chinchillas (p<0.001). FIG. 9B depicts representativeimages of biomasses that remained in the ears of animals immunized withadjuvant alone versus those immunized with IHF+adjuvant. Last column inFIG. 9B shows images of biomasses at the extremes of the scoring systemused here. Top image is that of a middle ear that contains a biomassthat would receive a score of 4+, whereas lower image is a healthymiddle ear that would receive a score of 0, indicating no biomass.

FIG. 10A shows H&E staining of a frozen section of a biofilm recoveredfrom the middle ear of a chinchilla that had been immunized via TCI withadjuvant alone versus immunization with IHF+adjuvant. Note condensed andcollapsed appearance of biofilm recovered from the animal immunized withIHF+adjuvant compared to that immunized with adjuvant alone. Imagesshown in FIG. 10A are shown at identical magnifications to illustratethe differences in height and density between the two representativebiomasses. FIG. 10B demonstrates that there is a significant reductionof bacterial load present in the middle ears of animals immunized withadjuvant alone versus those immunized with IHF+adjuvant (p<0.05).

FIG. 11 depicts an electrophoretic mobility shift assay whichdemonstrated that IHF formed specific complexes with dsDNA underconditions used to immunize chinchillas.

FIG. 12 graphically represents transcutaneous immunization with nativeIHF+adjuvant induced the formation of antibodies that significantlyreduced the biomass resident within the middle ears of chinchillascompared to receipt of either adjuvant alone (p<0.017), dsDNA alone(p<0.003) or IHF to which dsDNA was already bound+adjuvant (p<0.001).This outcome suggested that binding of dsDNA to native IHF maskedprotective epitopes of this DNABII family member.

FIG. 13 depicts the recognition of IHF (arrows) by antibody in serumafter subcutaneous immunization with IHF or with IHF to which dsDNA wasbound.’

FIGS. 14A-14C show a demonstration of synergism between a sub-optimalconcentration of anti-IHF serum (1:200) and DNAseI individually, thenmixed (FIG. 14A); a sub-optimal concentration of anti-IHF (1:100) andanti-outer membrane protein P5 serum (OMP P5) individually, then mixed(FIG. 14B); or that of an effective dilution of anti-IHF (in terms ofdebulking a biofilm but not inducing bacterial cell death) andamoxicillin individually, then mixed (FIG. 14C). In each of thesesituations, when any agent was combined with anti-IHF, the biofilmdebulking and/or killing effect observed was greater than that notedwhen any single agent was used alone. Note: biofilm height (in microns)is indicated under each image.

FIG. 15 is a comparison of E. coli treated with either naïve rabbitserum or rabbit anti-IHF serum (top row of images); with naive rat serumor rat anti-HNS serum (middle row of images); or with naive mouse serumor with mouse anti-DPS serum (bottom row of images). Note markedreduction in biomass following treatment with anti-IHF serum, howeverneither anti-FINS nor anti-DPS serum induced a reduction in biomass asused herein. Treatment with anti-IHF resulted in a 48.2% reduction inthe biofilm height, an 81% reduction in the biofilm average thickness,and a 64.5% reduction in the biomass. In contrast treatment with theanti-HNS or anti-DPS resulted in a nominal height reduction of 0.7% and4.2%, respectively, a reduction in the average thickness of 5.8% and6.4% respectively, and a reduction in the biomass of 0.3% and −17.4%respectively.

FIG. 16 shows in situ hybridization to demonstrate relative spatialdistribution of DNA from either a host organism or from the bacteriumwhen organized within a biofilm. In each circumstance, DNA appears asbrighter, whiter areas within the foreground of these black and whiteimages. DNA from the host is more densely labeled within the upper righthand image, demonstrating its more heavy distribution on the outerperiphery of the biofilm, whereas DNA from the bacterium is more denselylabeled in the lower left hand image of this 4-panel composite, therebydemonstrating its more dense distribution within the inner reaches ofthe biofilm.

FIG. 17 depicts the reduction of biofilms formed by NTHI upon incubationwith rabbit anti-IHF E. coli and chinchilla antibodies raised againstIhfA, fragment A5 and A3, compared to incubation with naïve rabbitserum, chinchilla serum, and sBHI.

FIG. 18 depicts the peptide sequences generated in the fine mappingprocess and highlights the peptides of interest based on epitope mappingusing chinchilla polyclonal antisera against either IHF from E. coliplus an adjuvant or IHF that has been complexed to an excess of DNA plusthe same adjuvant. Figure discloses SEQ ID NOS 6, 348-353, 341-347, 6,348-353 and 341-347, respectively, in order of appearance.

FIG. 19 is a sequence alignment of relevant portions of the DNA bindingproteins of various embodiments disclosed herein. Bold letters indicatean exact match to consensus, light gray lettering indicates aconservative amino acid change, and lightly or darkly shaded sequencesare highly conserved across species. Gray shaded undefined sequences atthe amino and/or carboxy-terminal are undefined amino acids that do notshare consensus sequences. FIG. 19 is based on information previouslypublished Oberto et al. (1994) Biochimie 76:901-908.

FIG. 20 is a comparison of the 16 amino acid peptide motif to Liu et al.(2008) Cell Microbiol. 10(1):262-276.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this disclosure belongs. All nucleotide sequencesprovided herein are presented in the 5′ to 3′ direction. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present disclosure,particular, non-limiting exemplary methods, devices, and materials arenow described. All technical and patent publications cited herein areincorporated herein by reference in their entirety. Nothing herein is tobe construed as an admission that the disclosure is not entitled toantedate such disclosure by virtue of prior invention.

The practice of the present disclosure will employ, unless otherwiseindicated, conventional techniques of tissue culture, immunology,molecular biology, microbiology, cell biology and recombinant DNA, whichare within the skill of the art. See, e.g., Sambrook and Russell eds,(2001) Molecular Cloning: A Laboratory Manual, 3^(rd) edition; theseries Ausubel et al. eds. (2007) Current Protocols in MolecularBiology; the series Methods in Enzymology (Academic Press, Inc., N.Y.);MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press atOxford University Press); MacPherson et al. (1995) PCR 2: A PracticalApproach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual;Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique,5^(th) edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No.4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization;Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds.(1984) Transcription and Translation; Immobilized Cells and Enzymes (IRLPress (1986)); Perbal (1984) A Practical Guide to Molecular Cloning;Miller and Calos eds, (1987) Gene Transfer Vectors for Mammalian Cells(Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer andExpression in Mammalian Cells; Mayer and Walker eds. (1987)Immunochemical Methods in Cell and Molecular Biology (Academic Press,London); and Herzenberg et al. eds (1996) Weir's Handbook ofExperimental Immunology.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 1.0 or 0.1, as appropriate oralternatively by a variation of +/−15%, or alternatively 10% oralternatively 5% or alternatively 2%. It is to be understood, althoughnot always explicitly stated, that all numerical designations arepreceded by the term “about”. It also is to be understood, although notalways explicitly stated, that the reagents described herein are merelyexemplary and that equivalents of such are known in the art.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a polypeptide” includes a plurality ofpolypeptides, including mixtures thereof.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination for the intended use. Thus, acomposition consisting essentially of the elements as defined hereinwould not exclude trace contaminants from the isolation and purificationmethod and pharmaceutically acceptable carriers, such as phosphatebuffered saline, preservatives, and the like. “Consisting of” shall meanexcluding more than trace elements of other ingredients and substantialmethod steps for administering the compositions disclosed herein.Embodiments defined by each of these transition terms are within thescope of this disclosure.

A “biofilm” intends an organized community of microorganisms that attimes adhere to the surface of a structure, that may be organic orinorganic, together with the polymers such as DNA that they secreteand/or release. The biofilms are very resistant to microbiotics andantimicrobial agents. They live on gingival tissues, teeth andrestorations, causing caries and periodontal disease, also known asperiodontal plaque disease. They also cause chronic middle earinfections. Biofilms can also form on the surface of dental implants,stents, catheter lines and contact lenses. They grow on pacemakers,heart valve replacements, artificial joints and other surgical implants.The Centers for Disease Control) estimate that over 65% of nosocomial(hospital-acquired) infections are caused by biofilms. They causechronic vaginal infections and lead to life-threatening systemicinfections in people with hobbled immune systems. Biofilms also areinvolved in numerous diseases. For instance, cystic fibrosis patientshave Pseudomonas infections that often result in antibiotic resistantbiofilms.

The term “inhibiting, competing or titrating” intends a reduction in theformation of the DNA/protein matrix (for example as shown in FIGS.1A-1C) that is a component of a microbial biofilm.

A “DNABII polypeptide or protein” intends a DNA binding protein orpolypeptide that is composed of DNA-binding domains and thus have aspecific or general affinity for microbial DNA. In one aspect, they bindDNA in the minor grove. Non-limiting examples of DNABII proteins are anintegration host factor (IHF) protein and a histone-like protein from E.coli strain U93 (HU). Other DNA binding proteins that may be associatedwith the biofilm include DPS (Genbank Accession No.: CAA49169), H-NS(Genbank Accession No.: CAA47740), Hfq (Genbank Accession No.:ACE63256), CbpA (Genbank Accession No.: BAA03950) and CbpB (GenbankAccession No.: NP-418813).

An “integration host factor” of “IHF” protein is a bacterial proteinthat is used by bacteriophages to incorporate their DNA into the hostbacteria. They also bind extracellular microbial DNA. The genes thatencode the IHF protein subunits in E. coli are himA (Genbank AccessionNo.: POA6X7.1) and himD (POA6Y1.1) genes. Homologs for these genes arefound in other organisms, and peptides corresponding to these genes fromother organisms can be found in Table 9.

“HMGB1” is a high mobility group box (HMGB) 1 protein that is reportedto bind to and distort the minor groove of DNA and is an example of aninterfering agent. Recombinant or isolated protein and polypeptide arecommercially available from Atgenglobal, ProSpecBio, Protein1 andAbnova.

“HU” or “histone-like protein from E. coli strain U93” refers to a classof heterodimeric proteins typically associate with E. coli. HU proteinsare known to bind DNA junctions. Related proteins have been isolatedfrom other microorganisms. The complete amino acid sequence of E. coliHU was reported by Laine et al. (1980) Eur. J. Biochem 103(3)447-481.Antibodies to the HU protein are commercially available from Abeam. Thegenes that encode the HU protein subunits in E. coli are hupA and hupBcorresponding to SEQ ID Nos: 28 and 29, respectively. Homologs for thesegenes are found in other organisms, and peptides corresponding to thesegenes from other organisms can be found in Table 9.

The term “surface antigens” or “surface proteins” refers to proteins orpeptides on the surface of cells such as bacterial cells. Examples ofsurface antigens are Outer membrane proteins such as OMP P5 (GenbankAccession No.: YP-004139079.1), OMP P2 (Genbank Accession No.:ZZX87199.1), OMP P26 (Genbank Accession No.: YP-665091.1), rsPilA orrecombinant soluble PilA (Genbank Accession No.: EFU96734.1) and Type IVPilin (Genbank Accession No.: Yp-003864351.1).

The term “Haemophilus influenzae” refers to pathogenic bacteria that cancause many different infections such as, for example, ear infections,eye infections, and sinusitis. Many different strains of Haemophilusinfluenzae have been isolated and have an IhfA gene or protein. Somenon-limiting examples of different strains of Haemophilus influenzaeinclude Rd KW20, 86-028NP, R2866, PittGG, PittEE, R2846, and 2019.

“Microbial DNA” intends single or double stranded DNA from amicroorganism that produces a biofilm.

“Inhibiting, preventing or breaking down” a biofilm intends theprophylactic or therapeutic reduction in the structure of a biofilm. Anexample of breaking down or reducing a biofilm is shown in FIG. 5 .

An “interfering agent” intends an agent that any one or more ofcompetes, inhibits, prevents, titrates a DNABII polypeptide such as IHFto a microbial DNA or also breaks down a microbial biofilm. It can beany one or more of a chemical or biological molecule. For example,DNABII such as IHF can specifically bind, bend or distorted DNAstructures such as DNA containing four-way junctions, cis-platinumadducts, DNA loop or base bulges. Examples of such agents, withoutlimitation, include (1) small molecules that inhibit the DNA-bindingactivity of IHF, (2) small molecules such as polyamines and sperminethat compete with IHF in DNA binding, (3) polypeptides such as peptidefragments of IHF that compete with IHF in DNA binding, (4) antibodies orfragments thereof directed to IHF, (5) a short polynucleotide that bindsthe polypeptide or biofilm forming DNA, or (6) a four-way or bentpolynucleotides or other types of polynucleotides containing bent ordistorted DNA structures that compete in IHF-binding. A “small moleculethat inhibits the binding of an IHF to a nucleic acid” refers to (1) or(2) above and includes those that bind DNA in the minor grove, i.e.,minor groove binding molecules. A “four-way polynucleotide” intends apolynucleotide that contains a four-way junction, also known as theHolliday junction, between four strands of DNA.

A “bent polynucleotide” intends a double strand polynucleotide thatcontains a small loop on one strand which does not pair with the otherstrand. In some embodiments, the loop is from 1 base to about 20 baseslong, or alternatively from 2 bases to about 15 bases long, oralternatively from about 3 bases to about 12 bases long, oralternatively from about 4 bases to about 10 bases long, oralternatively has about 4, 5, or 6, or 7, or 8, or 9, or 10 bases.

“Polypeptides that compete with DNABII binding, such as IHF in DNAbinding” intend proteins or peptides that compete with DNABII (e.g.,IHF) in binding bent or distorted DNA structures but do not form abiofilm with the DNA. Examples, without limitation, include fragments ofIHF that include one or more DNA binding domains of the IHF, or thebiological equivalents thereof. DNA binding domains are shown in FIGS.6A-6B.

A “subject” of diagnosis or treatment is a cell or an animal such as amammal, or a human. Non-human animals subject to diagnosis or treatmentand are those subject to infections or animal models, for example,simians, murines, such as, rats, mice, chinchilla, canine, such as dogs,leporids, such as rabbits, livestock, sport animals, and pets.

The term “protein”, “peptide” and “polypeptide” are used interchangeablyand in their broadest sense to refer to a compound of two or moresubunit amino acids, amino acid analogs or peptidomimetics. The subunitsmay be linked by peptide bonds. In another embodiment, the subunit maybe linked by other bonds, e.g., ester, ether, etc. A protein or peptidemust contain at least two amino acids and no limitation is placed on themaximum number of amino acids which may comprise a protein's orpeptide's sequence. As used herein the term “amino acid” refers toeither natural and/or unnatural or synthetic amino acids, includingglycine and both the D and L optical isomers, amino acid analogs andpeptidomimetics.

A “C-terminal polypeptide” intends at least the 10, or alternatively atleast the 15, or alternatively at least 20, or at least the 25C-terminal amino acids or alternatively half of a polypeptide. Inanother aspect, for polypeptides containing 90 amino acids, theC-terminal polypeptide would comprise amino acids 46 through 90. In oneaspect, the term intends the C-terminal 20 amino acids from the carboxyterminus.

A “tip fragment” of a DNABII polypeptide intends a DNA polypeptide that,using IHFalpha as an example, forms the two arms of the proteins (seeFIG. 6B). Non-limiting examples of such include IhfA, A tip fragment:NFELRDKSSRPGRNPKTGDVV (SEQ ID NO: 356) and IhfB, B tip fragment:SLHHRQPRLGRNPKTGDSVNL (SEQ ID NO: 357).

A “tail fragment” of a DNABII polypeptide intends a region of theprotein that is both exposed to the bulk medium and not occluded by DNAor other polypeptides.

An immunodominant antigen intends a region of the protein that isrecognized and binds with high affinity to an antibody.

An immunoprotective antigen intends a region of the protein that isrecognized and binds with high affinity to an antibody to interfere withprotein function; the antibodies generated against an immunoprotectiveantigen are characterized by enhanced or optimal effect against a targetindication as a result to the interference with protein function—in thiscase, an improve capability to clear biofilms.

The terms “polynucleotide” and “oligonucleotide” are usedinterchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides or analogsthereof. Polynucleotides can have any three-dimensional structure andmay perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: a gene or gene fragment (forexample, a probe, primer, EST or SAGE tag), exons, introns, messengerRNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes and primers. A polynucleotide can comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure can be impartedbefore or after assembly of the polynucleotide. The sequence ofnucleotides can be interrupted by non-nucleotide components. Apolynucleotide can be further modified after polymerization, such as byconjugation with a labeling component. The term also refers to bothdouble- and single-stranded molecules. Unless otherwise specified orrequired, any embodiment disclosed herein that is a polynucleotideencompasses both the double-stranded form and each of two complementarysingle-stranded forms known or predicted to make up the double-strandedform.

A polynucleotide is composed of a specific sequence of four nucleotidebases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil(U) for thymine when the polynucleotide is RNA. Thus, the term“polynucleotide sequence” is the alphabetical representation of apolynucleotide molecule. This alphabetical representation can be inputinto databases in a computer having a central processing unit and usedfor bioinformatics applications such as functional genomics and homologysearching.

The term “isolated” or “recombinant” as used herein with respect tonucleic acids, such as DNA or RNA, refers to molecules separated fromother DNAs or RNAs, respectively that are present in the natural sourceof the macromolecule as well as polypeptides. The term “isolated orrecombinant nucleic acid” is meant to include nucleic acid fragmentswhich are not naturally occurring as fragments and would not be found inthe natural state. The term “isolated” is also used herein to refer topolynucleotides, polypeptides and proteins that are isolated from othercellular proteins and is meant to encompass both purified andrecombinant polypeptides. In other embodiments, the term “isolated orrecombinant” means separated from constituents, cellular and otherwise,in which the cell, tissue, polynucleotide, peptide, polypeptide,protein, antibody or fragment(s) thereof, which are normally associatedin nature. For example, an isolated cell is a cell that is separatedfrom tissue or cells of dissimilar phenotype or genotype. An isolatedpolynucleotide is separated from the 3′ and 5′ contiguous nucleotideswith which it is normally associated in its native or naturalenvironment, e.g., on the chromosome. As is apparent to those of skillin the art, a non-naturally occurring polynucleotide, peptide,polypeptide, protein, antibody or fragment(s) thereof, does not require“isolation” to distinguish it from its naturally occurring counterpart.

It is to be inferred without explicit recitation and unless otherwiseintended, that when the present disclosure relates to a polypeptide,protein, polynucleotide or antibody, an equivalent or a biologicallyequivalent of such is intended within the scope of this disclosure. Asused herein, the term “biological equivalent thereof” is intended to besynonymous with “equivalent thereof” when referring to a referenceprotein, antibody, fragment, polypeptide or nucleic acid, intends thosehaving minimal homology while still maintaining desired structure orfunctionality. Unless specifically recited herein, it is contemplatedthat any polynucleotide, polypeptide or protein mentioned herein alsoincludes equivalents thereof. In one aspect, an equivalentpolynucleotide is one that hybridizes under stringent conditions to thepolynucleotide or complement of the polynucleotide as described hereinfor use in the described methods. In another aspect, an equivalentantibody or antigen binding polypeptide intends one that binds with atleast 70%, or alternatively at least 75%, or alternatively at least 80%,or alternatively at least 85%, or alternatively at least 90%, oralternatively at least 95% affinity or higher affinity to a referenceantibody or antigen binding fragment. In another aspect, the equivalentthereof competes with the binding of the antibody or antigen bindingfragment to its antigen tinder a competitive ELISA assay. In anotheraspect, an equivalent intends at least about 80% homology or identityand alternatively, at least about 85%, or alternatively at least about90%, or alternatively at least about 95%, or alternatively 98% percenthomology or identity and exhibits substantially equivalent biologicalactivity to the reference protein, polypeptide or nucleic acid. Examplesof biologically equivalent polypeptides are provided in Table 9 whichidentifies conservative amino acid substitutions to the disclosed aminoacid sequences.

A polynucleotide or polynucleotide region (or a polypeptide orpolypeptide region) having a certain percentage (for example, 80%, 85%,90%, or 95%) of “sequence identity” to another sequence means that, whenaligned, that percentage of bases (or amino acids) are the same incomparing the two sequences. The alignment and the percent homology orsequence identity can be determined using software programs known in theart, for example those described in Current Protocols in MolecularBiology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table7.7.1. In certain embodiments, default parameters are used foralignment. A non-limiting exemplary alignment program is BLAST, usingdefault parameters. In particular, exemplary programs include BLASTN andBLASTP, using the following default parameters: Genetic code=standard;filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Details of these programs can befound at the following Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST.Sequence identity and percent identity were determined by incorporatingthem into clustalW (available at the web address:align.genome.jp, lastaccessed on Mar. 7, 2011.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare homologous at that position. A degree of homology between sequencesis a function of the number of matching or homologous positions sharedby the sequences. An “unrelated” or “non-homologous” sequence sharesless than 40% identity, or alternatively less than 25% identity, withone of the sequences of the present disclosure.

“Homology” or “identity” or “similarity” can also refer to two nucleicacid molecules that hybridize under stringent conditions.

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson-Crick base pairing, Hoogstein binding, or inany other sequence-specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming amulti-stranded complex, a single self-hybridizing strand, or anycombination of these. A hybridization reaction may constitute a step ina more extensive process, such as the initiation of a PCR reaction, orthe enzymatic cleavage of a polynucleotide by a ribozyme.

Examples of stringent hybridization conditions include: incubationtemperatures of about 25° C. to about 37° C.; hybridization bufferconcentrations of about 6×SSC to about 10×SSC; formamide concentrationsof about 0% to about 25%; and wash solutions from about 4×SSC to about8×SSC. Examples of moderate hybridization conditions include: incubationtemperatures of about 40° C. to about 50° C.; buffer concentrations ofabout 9×SSC to about 2×SSC; formamide concentrations of about 30% toabout 50%; and wash solutions of about 5×SSC to about 2×SSC. Examples ofhigh stringency conditions include: incubation temperatures of about 55°C. to about 68° C.; buffer concentrations of about 1×SSC to about0.1×SSC; formamide concentrations of about 55% to about 75%; and washsolutions of about 1×SSC, 0.1×SSC, or deionized water. In general,hybridization incubation times are from 5 minutes to 24 hours, with 1,2, or more washing steps, and wash incubation times are about 1, 2, or15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It isunderstood that equivalents of SSC using other buffer systems can beemployed.

As used herein, “expression” refers to the process by whichpolynucleotides are transcribed into mRNA and/or the process by whichthe transcribed mRNA is subsequently being translated into peptides,polypeptides, or proteins. If the polynucleotide is derived from genomicDNA, expression may include splicing of the mRNA in a eukaryotic cell.

The term “encode” as it is applied to polynucleotides refers to apolynucleotide which is said to “encode” a polypeptide if, in its nativestate or when manipulated by methods well known to those skilled in theart, it can be transcribed and/or translated to produce the mRNA for thepolypeptide and/or a fragment thereof. The antisense strand is thecomplement of such a nucleic acid, and the encoding sequence can bededuced therefrom.

As used herein, the terms “treating,” “treatment” and the like are usedherein to mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disorder or sign or symptom thereof, and/or maybe therapeutic in terms of a partial or complete cure for a disorderand/or adverse effect attributable to the disorder.

To prevent intends to prevent a disorder or effect in vitro or in vivoin a system or subject that is predisposed to the disorder or effect. Anexample of such is preventing the formation of a biofilm in a systemthat is infected with a microorganism known to produce one.

A “composition” is intended to mean a combination of active agent andanother compound or composition, inert (for example, a detectable agentor label) or active, such as an adjuvant.

A “pharmaceutical composition” is intended to include the combination ofan active agent with a carrier, inert or active, making the compositionsuitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

“Pharmaceutically acceptable carriers” refers to any diluents,excipients, or carriers that may be used in the compositions disclosedherein. Pharmaceutically acceptable carriers include ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances, such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field. They may be selected with respect to the intendedform of administration, that is, oral tablets, capsules, elixirs, syrupsand the like, and consistent with conventional pharmaceutical practices.

A “biologically active agent” or an active agent disclosed hereinintends one or more of an isolated or recombinant polypeptide, anisolated or recombinant polynucleotide, a vector, an isolated host cell,or an antibody, as well as compositions comprising one or more of same.

“Administration” can be effected in one dose, continuously orintermittently throughout the course of treatment. Methods ofdetermining the most effective means and dosage of administration areknown to those of skill in the art and will vary with the compositionused for therapy, the purpose of the therapy, the target cell beingtreated, and the subject being treated. Single or multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician. Suitable dosage formulations andmethods of administering the agents are known in the art. Route ofadministration can also be determined and method of determining the mosteffective route of administration are known to those of skill in the artand will vary with the composition used for treatment, the purpose ofthe treatment, the health condition or disease stage of the subjectbeing treated, and target cell or tissue. Non-limiting examples of routeof administration include oral administration, nasal administration,injection, and topical application.

An agent of the present disclosure can be administered for therapy byany suitable route of administration. It will also be appreciated thatthe optimal route will vary with the condition and age of the recipient,and the disease being treated.

The term “effective amount” refers to a quantity sufficient to achieve adesired effect. In the context of therapeutic or prophylacticapplications, the effective amount will depend on the type and severityof the condition at issue and the characteristics of the individualsubject, such as general health, age, sex, body weight, and tolerance topharmaceutical compositions. In the context of an immunogeniccomposition, in some embodiments the effective amount is the amountsufficient to result in a protective response against a pathogen. Inother embodiments, the effective amount of an immunogenic composition isthe amount sufficient to result in antibody generation against theantigen. In some embodiments, the effective amount is the amountrequired to confer passive immunity on a subject in need thereof. Withrespect to immunogenic compositions, in some embodiments the effectiveamount will depend on the intended use, the degree of immunogenicity ofa particular antigenic compound, and the health/responsiveness of thesubject's immune system, in addition to the factors described above. Theskilled artisan will be able to determine appropriate amounts dependingon these and other factors.

In the case of an in vitro application, in some embodiments theeffective amount will depend on the size and nature of the applicationin question. It will also depend on the nature and sensitivity of the invitro target and the methods in use. The skilled artisan will be able todetermine the effective amount based on these and other considerations.The effective amount may comprise one or more administrations of acomposition depending on the embodiment.

The term “conjugated moiety” refers to a moiety that can be added to anisolated chimeric polypeptide by forming a covalent bond with a residueof chimeric polypeptide. The moiety may bond directly to a residue ofthe chimeric polypeptide or may form a covalent bond with a linker whichin turn forms a covalent bond with a residue of the chimericpolypeptide.

A “peptide conjugate” refers to the association by covalent ornon-covalent bonding of one or more polypeptides and another chemical orbiological compound. In a non-limiting example, the “conjugation” of apolypeptide with a chemical compound results in improved stability orefficacy of the polypeptide for its intended purpose. In one embodiment,a peptide is conjugated to a carrier, wherein the carrier is a liposome,a micelle, or a pharmaceutically acceptable polymer.

“Liposomes” are microscopic vesicles consisting of concentric lipidbilayers. Structurally, liposomes range in size and shape from longtubes to spheres, with dimensions from a few hundred Angstroms tofractions of a millimeter. Vesicle-forming lipids are selected toachieve a specified degree of fluidity or rigidity of the final complexproviding the lipid composition of the outer layer. These are neutral(cholesterol) or bipolar and include phospholipids, such asphosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidylinositol (PI), and sphingomyelin (SM) and other types ofbipolar lipids including but not limited todioleoylphosphatidylethanolamine (DOPE), with a hydrocarbon chain lengthin the range of 14-22, and saturated or with one or more double C═Cbonds. Examples of lipids capable of producing a stable liposome, alone,or in combination with other lipid components are phospholipids, such ashydrogenated soy phosphatidylcholine (HSPC), lecithin,phosphatidylethanolamine, lysolecithin, lysophosphatidylethanol-amine,phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin,cardiolipin, phosphatidic acid, cerebrosides,distearoylphosphatidylethan-olamine (DSPE), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),palmitoyloteoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE) anddioleoylphosphatidylethanolamine4-(N-maleimido-triethyl)cyclohexane-1-carboxylate (DOPE-mal). Additionalnon-phosphorous containing lipids that can become incorporated intoliposomes include stearylamine, dodecylamine, hexadecylamine, isopropylmyristate, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, acetylpalmitate, glycerol ricinoleate, hexadecyl stearate, amphoteric acrylicpolymers, polyethyloxylated fatty acid amides, and the cationic lipidsmentioned above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA,DPTAP, DSTAP, DC-Chol).

Negatively charged lipids include phosphatidic acid (PA),dipalmitoylphosphatidylglycerol (DPPG), dioteoylphosphatidylglycerol and(DOPG), dicetylphosphate that are able to form vesicles. Typically,liposomes can be divided into three categories based on their overallsize and the nature of the lamellar structure. The threeclassifications, as developed by the New York Academy Sciences Meeting,“Liposomes and Their Use in Biology and Medicine,” December 1977, aremulti-lamellar vesicles (MLVs), small uni-lamellar vesicles (SUVs) andlarge uni-lamellar vesicles (LUVs). The biological active agents can beencapsulated in such for administration in accordance with the methodsdescribed herein.

A “micelle” is an aggregate of surfactant molecules dispersed in aliquid colloid. A typical micelle in aqueous solution forms an aggregatewith the hydrophilic “head” regions in contact with surrounding solvent,sequestering the hydrophobic tail regions in the micelle center. Thistype of micelle is known as a normal phase micelle (oil-in-watermicelle). Inverse micelles have the head groups at the center with thetails extending out (water-in-oil micelle). Micelles can be used toattach a polynucleotide, polypeptide, antibody or composition describedherein to facilitate efficient delivery to the target cell or tissue.

The phrase “pharmaceutically acceptable polymer” refers to the group ofcompounds which can be conjugated to one or more polypeptides describedhere. It is contemplated that the conjugation of a polymer to thepolypeptide is capable of extending the half-life of the polypeptide invivo and in vitro. Non-limiting examples include polyethylene glycols,polyvinylpyrrolidones, polyvinylalcohols, cellulose derivatives,polyacrylates, polymethacrylates, sugars, polyols and mixtures thereof.The biological active agents can be conjugated to a pharmaceuticallyacceptable polymer for administration in accordance with the methodsdescribed herein.

A “gene delivery vehicle” is defined as any molecule that can carryinserted polynucleotides into a host cell. Examples of gene deliveryvehicles are liposomes, micelles biocompatible polymers, includingnatural polymers and synthetic polymers; lipoproteins; polypeptides;polysaccharides; lipopolysaccharides; artificial viral envelopes; metalparticles; and bacteria, or viruses, such as baculovirus, adenovirus andretrovirus, bacteriophage, cosmid, plasmid, fungal vectors and otherrecombination vehicles typically used in the art which have beendescribed for expression in a variety of eukaryotic and prokaryotichosts, and may be used for gene therapy as well as for simple proteinexpression.

A polynucleotide disclosed herein can be delivered to a cell or tissueusing a gene delivery vehicle. “Gene delivery,” “gene transfer,”“transducing,” and the like as used herein, are terms referring to theintroduction of an exogenous polynucleotide (sometimes referred to as a“transgene”) into a host cell, irrespective of the method used for theintroduction. Such methods include a variety of well-known techniquessuch as vector-mediated gene transfer (by, e.g., viralinfection/transfection, or various other protein-based or lipid-basedgene delivery complexes) as well as techniques facilitating the deliveryof “naked” polynucleotides (such as electroporation, “gene gun” deliveryand various other techniques used for the introduction ofpolynucleotides). The introduced polynucleotide may be stably ortransiently maintained in the host cell. Stable maintenance typicallyrequires that the introduced polynucleotide either contains an origin ofreplication compatible with the host cell or integrates into a repliconof the host cell such as an extrachromosomal replicon (e.g., a plasmid)or a nuclear or mitochondrial chromosome. A number of vectors are knownto be capable of mediating transfer of genes to mammalian cells, as isknown in the art and described herein.

As used herein the term “eDNA” refers to extracellular DNA found as acomponent to pathogenic biofilms.

A “plasmid” is an extra-chromosomal DNA molecule separate from thechromosomal DNA which is capable of replicating independently of thechromosomal DNA. In many cases, it is circular and double-stranded.Plasmids provide a mechanism for horizontal gene transfer within apopulation of microbes and typically provide a selective advantage undera given environmental state. Plasmids may carry genes that provideresistance to naturally occurring antibiotics in a competitiveenvironmental niche, or alternatively the proteins produced may act astoxins under similar circumstances.

“Plasmids” used in genetic engineering are called “plasmid vectors”.Many plasmids are commercially available for such uses. The gene to bereplicated is inserted into copies of a plasmid containing genes thatmake cells resistant to particular antibiotics and a multiple cloningsite (MCS, or polylinker), which is a short region containing severalcommonly used restriction sites allowing the easy insertion of DNAfragments at this location. Another major use of plasmids is to makelarge amounts of proteins. In this case, researchers grow bacteriacontaining a plasmid harboring the gene of interest. Just as thebacterium produces proteins to confer its antibiotic resistance, it canalso be induced to produce large amounts of proteins from the insertedgene. This is a cheap and easy way of mass-producing a gene or theprotein it then codes for.

A “yeast artificial chromosome” or “YAC” refers to a vector used toclone large DNA fragments (larger than 100 kb and up to 3000 kb). It isan artificially constructed chromosome and contains the telomeric,centromeric, and replication origin sequences needed for replication andpreservation in yeast cells. Built using an initial circular plasmid,they are linearized by using restriction enzymes, and then DNA ligasecan add a sequence or gene of interest within the linear molecule by theuse of cohesive ends. Yeast expression vectors, such as YACs, YIps(yeast integrating plasmid), and YEps (yeast episomal plasmid), areextremely useful as one can get eukaryotic protein products withposttranslational modifications as yeasts are themselves eukaryoticcells, however YACs have been found to be more unstable than BACs,producing chimeric effects.

A “viral vector” is defined as a recombinantly produced virus or viralparticle that comprises a polynucleotide to be delivered into a hostcell, either in vivo, ex vivo or in vitro. Examples of viral vectorsinclude retroviral vectors, adenovirus vectors, adeno-associated virusvectors, alphavirus vectors and the like. Infectious tobacco mosaicvirus (TMV)-based vectors can be used to manufacturer proteins and havebeen reported to express Griffithsin in tobacco leaves (O'Keefe et al.(2009) Proc. Nat. Acad. Sci. USA 106(15):6099-6104). Alphavirus vectors,such as Semliki Forest virus-based vectors and Sindbis virus-basedvectors, have also been developed for use in gene therapy andimmunotherapy. See, Schlesinger & Dubensky (1999) Curr. Opin.Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827. Inaspects where gene transfer is mediated by a retroviral vector, a vectorconstruct refers to the polynucleotide comprising the retroviral genomeor part thereof, and a therapeutic gene.

As used herein, “retroviral mediated gene transfer” or “retroviraltransduction” carries the same meaning and refers to the process bywhich a gene or nucleic acid sequences are stably transferred into thehost cell by virtue of the virus entering the cell and integrating itsgenome into the host cell genome. The virus can enter the host cell viaits normal mechanism of infection or be modified such that it binds to adifferent host cell surface receptor or ligand to enter the cell. Asused herein, retroviral vector refers to a viral particle capable ofintroducing exogenous nucleic acid into a cell through a viral orviral-like entry mechanism.

Retroviruses carry their genetic information in the form of RNA;however, once the virus infects a cell, the RNA is reverse-transcribedinto the DNA form which integrates into the genomic DNA of the infectedcell. The integrated DNA form is called a provirus.

In aspects where gene transfer is mediated by a DNA viral vector, suchas an adenovirus (Ad) or adeno-associated virus (AAV), a vectorconstruct refers to the polynucleotide comprising the viral genome orpart thereof, and a transgene. Adenoviruses (Ads) are a relatively wellcharacterized, homogenous group of viruses, including over 50 serotypes.See, e.g., PCT International Application Publication No. WO 95/27071.Ads do not require integration into the host cell genome. Recombinant Adderived vectors, particularly those that reduce the potential forrecombination and generation of wild-type virus, have also beenconstructed. See, PCT International Application Publication Nos. WO95/00655 and WO 95/11984, Wild-type AAV has high infectivity andspecificity integrating into the host cell's genome. See, Hermonat &Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470 and Lebkowski etal. (1988) Mol. Cell. Biol. 8:3988-3996.

Vectors that contain both a promoter and a cloning site into which apolynucleotide can be operatively linked are well known in the art. Suchvectors are capable of transcribing RNA in vitro or in vivo, and arecommercially available from sources such as Stratagene (La Jolla,Calif.) and Promega Biotech (Madison, Wis.). In order to optimizeexpression and/or in vitro transcription, it may be necessary to remove,add or alter 5′ and/or 3′ untranslated portions of the clones toeliminate extra, potential inappropriate alternative translationinitiation codons or other sequences that may interfere with or reduceexpression, either at the level of transcription or translation.Alternatively, consensus ribosome binding sites can be insertedimmediately 5′ of the start codon to enhance expression.

Gene delivery vehicles also include DNA/liposome complexes, micelles andtargeted viral protein-DNA complexes. Liposomes that also comprise atargeting antibody or fragment thereof can be used in the methodsdisclosed herein. In addition to the delivery of polynucleotides to acell or cell population, direct introduction of the proteins describedherein to the cell or cell population can be done by the non-limitingtechnique of protein transfection, alternatively culturing conditionsthat can enhance the expression and/or promote the activity of theproteins disclosed herein are other non-limiting techniques.

As used herein, the terms “antibody,” “antibodies” and “immunoglobulin”includes whole antibodies and any antigen binding fragment or a singlechain thereof. Thus the term “antibody” includes any protein or peptidecontaining molecule that comprises at least a portion of animmunoglobulin molecule. The terms “antibody,” “antibodies” and“immunoglobulin” also include immunoglobulins of any isotype, fragmentsof antibodies which retain specific binding to antigen, including, butnot limited to, Fab, Fab′, F(ab)₂, Fv, scFv, dsFv, Fd fragments, dAb,VH, VL, VhH, and V-NAR domains; minibodies, diabodies, triabodies,tetrabodies and kappa bodies; multispecific antibody fragments formedfrom antibody fragments and one or more isolated. Examples of suchinclude, but are not limited to a complementarity determining region(CDR) of a heavy or light chain or a ligand binding portion thereof, aheavy chain or light chain variable region, a heavy chain or light chainconstant region, a framework (FR) region, or any portion thereof, atleast one portion of a binding protein, chimeric antibodies, humanizedantibodies, single-chain antibodies, and fusion proteins comprising anantigen-binding portion of an antibody and a non-antibody protein. Thevariable regions of the heavy and light chains of the immunoglobulinmolecule contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies (Abs) may mediate the binding of theimmunoglobulin to host tissues. The term “anti-” when used before aprotein name, anti-DNABII, anti-IHF, anti-HU, anti-OMP P5, for example,refers to a monoclonal or polyclonal antibody that binds and/or has anaffinity to a particular protein. For example, “anti-IHF” refers to anantibody that binds to the IHF protein. The specific antibody may haveaffinity or bind to proteins other than the protein it was raisedagainst. For example, anti-IHF, while specifically raised against theIHF protein, may also bind other proteins that are related eitherthrough sequence homology or through structure homology.

The antibodies can be polyclonal, monoclonal, multispecific (e.g.,bispecific antibodies), and antibody fragments, so long as they exhibitthe desired biological activity. Antibodies can be isolated from anysuitable biological source, e.g., murine, rat, sheep and canine.

As used herein, “monoclonal antibody” refers to an antibody obtainedfrom a substantially homogeneous antibody population. Monoclonalantibodies are highly specific, as each monoclonal antibody is directedagainst a single determinant on the antigen. The antibodies may bedetectably labeled, e.g., with a radioisotope, an enzyme which generatesa detectable product, a fluorescent protein, and the like. Theantibodies may be further conjugated to other moieties, such as membersof specific binding pairs, e.g., biotin (member of biotin-avidinspecific binding pair), and the like. The antibodies may also be boundto a solid support, including, but not limited to, polystyrene plates orbeads, and the like.

Monoclonal antibodies may be generated using hybridoma techniques orrecombinant DNA methods known in the art. A hybridoma is a cell that isproduced in the laboratory from the fusion of an antibody-producinglymphocyte and a non-antibody producing cancer cell, usually a myelomaor lymphoma. A hybridoma proliferates and produces a continuous sampleof a specific monoclonal antibody. Alternative techniques for generatingor selecting antibodies include in vitro exposure of lymphocytes toantigens of interest, and screening of antibody display libraries incells, phage, or similar systems.

The term “human antibody” as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies disclosed hereinmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody” as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences. Thus, as used herein, the term “human antibody”refers to an antibody in which substantially every part of the protein(e.g., CDR, framework, C_(L), C_(H) domains (e.g., C_(H1), C_(H2),C_(H3)), hinge, (VL, VH)) is substantially non-immunogenic in humans,with only minor sequence changes or variations. Similarly, antibodiesdesignated primate (monkey, baboon, chimpanzee, etc.), rodent (mouse,rat, rabbit, guinea pig, hamster, and the like) and other mammalsdesignate such species, sub-genus, genus, sub-family, family specificantibodies. Further, chimeric antibodies include any combination of theabove. Such changes or variations optionally retain or reduce theimmunogenicity in humans or other species relative to non-modifiedantibodies. Thus, a human antibody is distinct from a chimeric orhumanized antibody. It is pointed out that a human antibody can beproduced by a non-human animal or prokaryotic or eukaryotic cell that iscapable of expressing functionally rearranged human immunoglobulin(e.g., heavy chain and/or light chain) genes. Further, when a humanantibody is a single chain antibody, it can comprise a linker peptidethat is not found in native human antibodies. For example, an Fv cancomprise a linker peptide, such as two to about eight glycine or otheramino acid residues, which connects the variable region of the heavychain and the variable region of the light chain. Such linker peptidesare considered to be of human origin.

As used herein, a human antibody is “derived from” a particular germlinesequence if the antibody is obtained from a system using humanimmunoglobulin sequences, e.g., by immunizing a transgenic mousecarrying human immunoglobulin genes or by screening a humanimmunoglobulin gene library. A human antibody that is “derived from” ahuman germline immunoglobulin sequence can be identified as such bycomparing the amino acid sequence of the human antibody to the aminoacid sequence of human germline immunoglobulins. A selected humanantibody typically is at least 90% identical in amino acids sequence toan amino acid sequence encoded by a human germline immunoglobulin geneand contains amino acid residues that identify the human antibody asbeing human when compared to the germline immunoglobulin amino acidsequences of other species (e.g., murine germline sequences). In certaincases, a human antibody may be at least 95%, or even at least 96%, 97%,98%, or 99% identical in amino acid sequence to the amino acid sequenceencoded by the germline immunoglobulin gene. Typically, a human antibodyderived from a particular human germline sequence will display no morethan 10 amino acid differences, from the amino acid sequence encoded bythe human germline immunoglobulin gene. In certain cases, the humanantibody may display no more than 5, or even no more than 4, 3, 2, or 1amino acid difference from the amino acid sequence encoded by thegermline immunoglobulin gene.

A “human monoclonal antibody” refers to antibodies displaying a singlebinding specificity which have variable and constant regions derivedfrom human germline immunoglobulin sequences. The term also intendsrecombinant human antibodies. Methods to making these antibodies aredescribed herein.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal (e.g., amouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the antibody, e.g., from a transfectoma,antibodies isolated from a recombinant, combinatorial human antibodylibrary, and antibodies prepared, expressed, created or isolated by anyother means that involve splicing of human immunoglobulin gene sequencesto other DNA sequences. Such recombinant human antibodies have variableand constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo. Methods to makingthese antibodies are described herein.

As used herein, chimeric antibodies are antibodies whose light and heavychain genes have been constructed, typically by genetic engineering,from antibody variable and constant region genes belonging to differentspecies.

As used herein, the term “humanized antibody” or “humanizedimmunoglobulin” refers to a human/non-human chimeric antibody thatcontains a minimal sequence derived from non-human immunoglobulin. Forthe most part, humanized antibodies are human immunoglobulins (recipientantibody) in which residues from a variable region of the recipient arereplaced by residues from a variable region of a non-human species(donor antibody) such as mouse, rat, rabbit, or non-human primate havingthe desired specificity, affinity and capacity. Humanized antibodies maycomprise residues that are not found in the recipient antibody or in thedonor antibody. The humanized antibody can optionally also comprise atleast a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin, a non-human antibody containing one ormore amino acids in a framework region, a constant region or a CDR, thathave been substituted with a correspondingly positioned amino acid froma human antibody. In general, humanized antibodies are expected toproduce a reduced immune response in a human host, as compared to anon-humanized version of the same antibody. The humanized antibodies mayhave conservative amino acid substitutions which have substantially noeffect on antigen binding or other antibody functions. Conservativesubstitutions groupings include: glycine-alanine,valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, serine-threonine and asparagine-glutamine.

The terms “polyclonal antibody” or “polyclonal antibody composition” asused herein refer to a preparation of antibodies that are derived fromdifferent B-cell lines. They are a mixture of immunoglobulin moleculessecreted against a specific antigen, each recognizing a differentepitope.

As used herein, the term “antibody derivative”, comprises a full-lengthantibody or a fragment of an antibody, wherein one or more of the aminoacids are chemically modified by alkylation, pegylation, acylation,ester formation or amide formation or the like, e.g., for linking theantibody to a second molecule. This includes, but is not limited to,pegylated antibodies, cysteine-pegylated antibodies, and variantsthereof.

As used herein, the term “label” intends a directly or indirectlydetectable compound or composition that is conjugated directly orindirectly to the composition to be detected, e.g., N-terminal histidinetags (N-His), magnetically active isotopes, e.g., ¹¹⁵Sn, ¹¹⁷Sn and¹¹⁹Sn, a nonradioactive isotopes such as ¹³C and ¹⁵N, polynucleotide orprotein such as an antibody so as to generate a “labeled” composition.The term also includes sequences conjugated to the polynucleotide thatwill provide a signal upon expression of the inserted sequences, such asgreen fluorescent protein (GFP) and the like. The label may bedetectable by itself (e.g., radioisotope labels or fluorescent labels)or, in the case of an enzymatic label, may catalyze chemical alterationof a substrate compound or composition which is detectable. The labelscan be suitable for small scale detection or more suitable forhigh-throughput screening. As such, suitable labels include, but are notlimited to magnetically active isotopes, non-radioactive isotopes,radioisotopes, fluorochromes, chemiluminescent compounds, dyes, andproteins, including enzymes. The label may be simply detected or it maybe quantified. A response that is simply detected generally comprises aresponse whose existence merely is confirmed, whereas a response that isquantified generally comprises a response having a quantifiable (e.g.,numerically reportable) value such as an intensity, polarization, and/orother property. In luminescence or fluorescence assays, the detectableresponse may be generated directly using a luminophore or fluorophoreassociated with an assay component actually involved in binding, orindirectly using a luminophore or fluorophore associated with another(e.g., reporter or indicator) component. Examples of luminescent labelsthat produce signals include, but are not limited to bioluminescence andchemiluminescence. Detectable luminescence response generally comprisesa change in, or an occurrence of a luminescence signal. Suitable methodsand luminophores for luminescently labeling assay components are knownin the art and described for example in Haugland, Richard P. (1996)Handbook of Fluorescent Probes and Research Chemicals (6^(th) ed).Examples of luminescent probes include, but are not limited to, aequorinand luciferases.

As used herein, the term “immunoconjugate” comprises an antibody or anantibody derivative associated with or linked to a second agent, such asa cytotoxic agent, a detectable agent, a radioactive agent, a targetingagent, a human antibody, a humanized antibody, a chimeric antibody, asynthetic antibody, a semisynthetic antibody, or a multispecificantibody.

Examples of suitable fluorescent labels include, but are not limited to,fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin,coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, LuciferYellow, Cascade Blue™, and Texas Red. Other suitable optical dyes aredescribed in the Haugland, Richard P. (1996) Handbook of FluorescentProbes and Research Chemicals (6^(th) ed.).

In another aspect, the fluorescent label is functionalized to facilitatecovalent attachment to a cellular component present in or on the surfaceof the cell or tissue such as a cell surface marker. Suitable functionalgroups, include, but are not limited to, isothiocyanate groups, aminogroups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonylhalides, all of which may be used to attach the fluorescent label to asecond molecule. The choice of the functional group of the fluorescentlabel will depend on the site of attachment to either a linker, theagent, the marker, or the second labeling agent.

“Eukaryotic cells” comprise all of the life kingdoms except monera. Theycan be easily distinguished through a membrane-bound nucleus. Animals,plants, fungi, and protists are eukaryotes or organisms whose cells areorganized into complex structures by internal membranes and acytoskeleton. The most characteristic membrane-bound structure is thenucleus. Unless specifically recited, the term “host” includes aeukaryotic host, including, for example, yeast, higher plant, insect andmammalian cells. Non-limiting examples of eukaryotic cells or hostsinclude simian, bovine, porcine, murine, rat, avian, reptilian andhuman.

“Prokaryotic cells” that usually lack a nucleus or any othermembrane-bound organelles and are divided into two domains, bacteria andarchaea. In addition to chromosomal DNA, these cells can also containgenetic information in a circular loop called on episome. Bacterialcells are very small, roughly the size of an animal mitochondrion (about1-2 μm in diameter and 10 μm long). Prokaryotic cells feature threemajor shapes: rod shaped, spherical, and spiral. Instead of goingthrough elaborate replication processes like eukaryotes, bacterial cellsdivide by binary fission. Examples include but are not limited toBacillus bacteria, E. coli bacterium, and Salmonella bacterium.

A “native” or “natural” antigen is a polypeptide, protein or a fragmentwhich contains an epitope, which has been isolated from a naturalbiological source, and which can specifically bind to an antigenreceptor, in particular a T cell antigen receptor (TCR), in a subject.

The terms “antigen” and “antigenic” refer to molecules with the capacityto be recognized by an antibody or otherwise act as a member of anantibody-ligand pair. “Specific binding” refers to the interaction of anantigen with the variable regions of immunoglobulin heavy and lightchains. Antibody-antigen binding may occur in vivo or in vitro. Theskilled artisan will understand that macromolecules, including proteins,nucleic acids, fatty acids, lipids, lipopolysaccharides andpolysaccharides have the potential to act as an antigen. The skilledartisan will further understand that nucleic acids encoding a proteinwith the potential to act as an antibody ligand necessarily encode anantigen. The artisan will further understand that antigens are notlimited to full-length molecules, but can also include partialmolecules. The term “antigenic” is an adjectival reference to moleculeshaving the properties of an antigen. The term encompasses substanceswhich are immunogenic, i.e., immunogens, as well as substances whichinduce immunological unresponsiveness, or anergy, i.e., anergens.

An “altered antigen” is one having a primary sequence that is differentfrom that of the corresponding wild-type antigen. Altered antigens canbe made by synthetic or recombinant methods and include, but are notlimited to, antigenic peptides that are differentially modified duringor after translation, e.g., by phosphorylation, glycosylation,cross-linking, acylation, proteolytic cleavage, linkage to an antibodymolecule, membrane molecule or other ligand. (Ferguson et al. (1988)Ann. Rev. Biochem. 57:285-320). A synthetic or altered antigen disclosedherein is intended to bind to the same TCR as the natural epitope.

A “self-antigen” also referred to herein as a native or wild-typeantigen is an antigenic peptide that induces little or no immuneresponse in the subject due to self-tolerance to the antigen. An exampleof a self-antigen is the melanoma specific antigen gp100.

The terms “major histocompatibility complex” or “MHC” refers to acomplex of genes encoding cell-surface molecules that are required forantigen presentation to T cells and for rapid graft rejection. Inhumans, the MI-IC is also known as the “human leukocyte antigen” or“HLA” complex. The proteins encoded by the MHC are known as “MHCmolecules” and are classified into class I and class II MHC molecules.Class I MHC includes membrane heterodimeric proteins made up of a chainencoded in the MHC noncovalently linked with the β2-microglobulin. ClassI MHC molecules are expressed by nearly all nucleated cells and havebeen shown to function in antigen presentation to CD8⁺ T cells. Class Imolecules include HLA-A, B, and C in humans. Class II MHC molecules alsoinclude membrane heterodimeric proteins consisting of noncovalentlyassociated α and β chains. Class II MHC molecules are known to functionin CD4⁻ T cells and, in humans, include HLA-DP, -DQ, and DR. In aparticular embodiment, compositions and ligands can complex with MHCmolecules of any HLA type. Those of skill in the art are familiar withthe serotypes and genotypes of the HLA. See:bimas.dcrt.nih.gov/cgi-bin/molbio/hla coefficient viewing page.Rammensee H. G., Bachmann J., and Stevanovic S. MHC Ligands and PeptideMotifs (1997) Chapman & Hall Publishers; Schreuder G. M. Th. et al. TheHLA dictionary (1999) Tissue Antigens 54:409-437.

The term “antigen-presenting matrix”, as used herein, intends a moleculeor molecules which can present antigen in such a way that the antigencan be bound by a T-cell antigen receptor on the surface of a T cell. Anantigen-presenting matrix can be on the surface of an antigen-presentingcell (APC), on a vesicle preparation of an APC, or can be in the form ofa synthetic matrix on a solid support such as a bead or a plate. Anexample of a synthetic antigen-presenting matrix is purified MHC class Imolecules complexed to P2-microglobulin, multimers of such purified MHCclass I molecules, purified MI-IC Class II molecules, or functionalportions thereof, attached to a solid support.

The term “antigen presenting cells (APC)” refers to a class of cellscapable of presenting one or more antigens in the form of antigen-MHCcomplex recognizable by specific effector cells of the immune system,and thereby inducing an effective cellular immune response against theantigen or antigens being presented. While many types of cells may becapable of presenting antigens on their cell surface for T-cellrecognition, only professional APCs have the capacity to presentantigens in an efficient amount and further to activate T-cells forcytotoxic T-lymphocyte (CTL) responses. APCs can be intact whole cellssuch as macrophages, B-cells and dendritic cells; or other molecules,naturally occurring or synthetic, such as purified MHC class moleculescomplexed to β2-microglobulin.

The term “dendritic cells (DCs)” refers to a diverse population ofmorphologically similar cell types found in a variety of lymphoid andnon-lymphoid tissues (Steinman (1991) Ann. Rev. Immunol. 9:271-296).Dendritic cells constitute the most potent mammalian APCs. A subset, ifnot all, of DCs are derived from bone marrow progenitor cells, circulatein small numbers in the peripheral blood and appear either as immatureLangerhans' cells or terminally differentiated mature cells. While DCscan be derived from monocytes, they possess distinct phenotypes. Forexample, a particular differentiating marker, CD14 antigen, is not foundin dendritic cells but is expressed at very high levels in monocytes bymonocytes. See for example Jersmann et al. (2005) Immunol. Cell Biol.83:462.

Also, mature dendritic cells are not phagocytic, whereas the monocytesare strongly phagocytosing cells. Mature monocytes and DCs endocytosematerial through different mechanisms. Monocytes engulf by means ofphagocytosis, whereas DCs utilize macropinocytosis. Thus, DCs generallyengulf cargo of a smaller size than monocytes (See for example Connerand Schmid (2003) Nature 433:37-44. It has been shown that DCs are asendocytically active as other antigen presenting cells, and provide allthe signals necessary for T cell activation and proliferation (See,e.g., Levine and Chain (1992) PNAS 89(17):8342.

The term “antigen presenting cell recruitment factors” or “APCrecruitment factors” include both intact, whole cells as well as othermolecules that are capable of recruiting antigen presenting cells.Examples of suitable APC recruitment factors include molecules such asinterleukin 4 (IL4), granulocyte macrophage colony stimulating factor(GM-CSF), Sepragel and macrophage inflammatory protein 3 alpha (MIP3α).These are available from Immunex, Schering-Plough and R&D Systems(Minneapolis, Minn.). They also can be recombinantly produced using themethods disclosed in Current Protocols In Molecular Biology (F. M.Ausubel et al., eds. (1987)). Peptides, proteins and compounds havingthe same biological activity as the above-noted factors are includedwithin the scope of this disclosure.

The term “immune effector cells” refers to cells capable of binding anantigen and which mediate an immune response. These cells include, butare not limited to, T cells, B cells, monocytes, macrophages, NK cellsand cytotoxic T lymphocytes (CTLs), for example CTL lines, CTL clones,and CTLs from tumor, inflammatory, or other infiltrates. Certaindiseased tissue expresses specific antigens and CTLs specific for theseantigens have been identified.

The term “immune effector molecule” as used herein, refers to moleculescapable of antigen-specific binding, and includes antibodies, T cellantigen receptors, B cell antigen receptors, and MI-IC Class I and ClassII molecules.

A “naive” immune effector cell is an immune effector cell that has neverbeen exposed to an antigen capable of activating that cell. Activationof naive immune effector T cells requires both recognition of theantigen:MHC complex and the simultaneous delivery of a costimulatorysignal by a professional APC in order to proliferate and differentiateinto antigen-specific armed effector T cells. Activated T cells can thenactivate specific B cells through immunological synapses by providing aco-stimulation signal. Activated B cells subsequently produce antibodiesdirected to a specific antigen. Naïve B cells can also be activated by Tcell-independent mechanisms. This occurs when antigens are capable ofbinding to the B cell receptor and producing a co-stimulation signal.

“Immune response” broadly refers to the antigen-specific responses oflymphocytes to foreign substances. The terms “immunogen” and“immunogenic” refer to molecules with the capacity to elicit an immuneresponse. All immunogens are antigens, however, not all antigens areimmunogenic. An immune response disclosed herein can be humoral (viaantibody activity) or cell-mediated (via T cell activation). Theresponse may occur in vivo or in vitro. The skilled artisan willunderstand that a variety of macromolecules, including proteins, nucleicacids, fatty acids, lipids, lipopolysaccharides and polysaccharides havethe potential to be immunogenic. The skilled artisan will furtherunderstand that nucleic acids encoding a molecule capable of elicitingan immune response necessarily encode an immunogen. The artisan willfurther understand that immunogens are not limited to full-lengthmolecules, but may include partial molecules.

The term “passive immunity” refers to the transfer of immunity from onesubject to another through the transfer of antibodies. Passive immunitymay occur naturally, as when maternal antibodies are transferred to afetus. Passive immunity may also occur artificially as when antibodycompositions are administered to non-immune subjects. Antibody donorsand recipients may be human or non-human subjects. Antibodies may bepolyclonal or monoclonal, may be generated in vitro or in vivo, and maybe purified, partially purified, or unpurified depending on theembodiment. In some embodiments described herein, passive immunity isconferred on a subject in need thereof through the administration ofantibodies or antigen binding fragments that specifically recognize orbind to a particular antigen. In some embodiments, passive immunity isconferred through the administration of an isolated or recombinantpolynucleotide encoding an antibody or antigen binding fragment thatspecifically recognizes or binds to a particular antigen.

In the context of this disclosure, a “ligand” is a polypeptide. In oneaspect, the term “ligand” as used herein refers to any molecule thatbinds to a specific site on another molecule. In other words, the ligandconfers the specificity of the protein in a reaction with an immuneeffector cell or an antibody to a protein or DNA to a protein. In oneaspect it is the ligand site within the protein that combines directlywith the complementary binding site on the immune effector cell.

In one aspect, a peptide or ligand disclosed herein binds to anantigenic determinant or epitope on an immune effector cell, such as anantibody or a T cell receptor (TCR). A ligand may be an antigen,peptide, protein or epitope disclosed herein.

In another aspect, ligands may bind to a receptor on an antibody. In oneembodiment, the ligand disclosed herein is about 4 to about 8 aminoacids in length.

In a further aspect, ligands may bind to a receptor on an MHC class Imolecule. In one embodiment, the ligand disclosed herein is about 7 toabout 11 amino acids in length.

In a yet further aspect, ligands may bind to a receptor on an MHC classII molecule. In one embodiment, the ligand disclosed herein is about 10to about 20 amino acids long.

As used herein, the term “educated, antigen-specific immune effectorcell”, is an immune effector cell as defined above, which has previouslyencountered an antigen. In contrast with its naive counterpart,activation of an educated, antigen-specific immune effector cell doesnot require a costimulatory signal. Recognition of the peptide:MHCcomplex is sufficient.

“Activated”, when used in reference to a T cell, implies that the cellis no longer in Go phase, and begins to produce one or more ofcytotoxins, cytokines, and other related membrane-associated proteinscharacteristic of the cell type (e.g., CD8⁺ or CD4⁺), is capable ofrecognizing and binding any target cell that displays the particularantigen on its surface, and releasing its effector molecules.

The term “cross-reactive” is used to describe compounds disclosed hereinwhich are functionally overlapping. More particularly, the immunogenicproperties of a native ligand and/or immune effector cells activatedthereby are shared to a certain extent by the altered ligand such thatthe altered ligand is “cross-reactive” with the native ligand and/or theimmune effector cells activated thereby. For purposes of thisdisclosure, cross-reactivity is manifested at multiple levels: (i) atthe ligand level, e.g., the altered ligands can bind the TCR of andactivate native ligand CTLs; (ii) at the T cell level, i.e., alteredligands disclosed herein bind the TCR of and activate a population of Tcells (distinct from the population of native ligand CTLs) which caneffectively target and lyse cells displaying the native ligand; and(iii) at the antibody level, e.g., “anti”-altered ligand antibodies candetect, recognize and bind the native ligand and initiate effectormechanisms in an immune response which ultimately result in eliminationof the native ligand from the host.

As used herein, the term “inducing an immune response in a subject” is aterm well understood in the art and intends that an increase of at leastabout 2-fold, at least about 5-fold, at least about 10-fold, at leastabout 100-fold, at least about 500-fold, or at least about 1000-fold ormore in an immune response to an antigen (or epitope) can be detected ormeasured, after introducing the antigen (or epitope) into the subject,relative to the immune response (if any) before introduction of theantigen (or epitope) into the subject. An immune response to an antigen(or epitope), includes, but is not limited to, production of anantigen-specific (or epitope-specific) antibody, and production of animmune cell expressing on its surface a molecule which specificallybinds to an antigen (or epitope). Methods of determining whether animmune response to a given antigen (or epitope) has been induced arewell known in the art. For example, antigen-specific antibody can bedetected using any of a variety of immunoassays known in the art,including, but not limited to, ELISA, wherein, for example, binding ofan antibody in a sample to an immobilized antigen (or epitope) isdetected with a detectably-labeled second antibody (e.g., enzyme-labeledmouse anti-human Ig antibody).

“Co-stimulatory molecules” are involved in the interaction betweenreceptor-ligand pairs expressed on the surface of antigen presentingcells and T cells. Research accumulated over the past several years hasdemonstrated convincingly that resting T cells require at least twosignals for induction of cytokine gene expression and proliferation(Schwartz (1990) Science 248:1349-1356 and Jenkins (1992) Immunol. Today13:69-73). One signal, the one that confers specificity, can be producedby interaction of the TCR/CD3 complex with an appropriate MHC/peptidecomplex. The second signal is not antigen specific and is termed the“co-stimulatory” signal. This signal was originally defined as anactivity provided by bone-marrow-derived accessory cells such asmacrophages and dendritic cells, the so called “professional” APCs.Several molecules have been shown to enhance co-stimulatory activity.These are heat stable antigen (HSA) (Liu et al. (1992) J. Exp. Med.175:437-445), chondroitin sulfate-modified MHC invariant chain (Ii-CS)(Naujokas et al. (1993) Cell 74:257-268), intracellular adhesionmolecule 1 (ICAM-1) (Van (1992) Cell 71:1065-1068). These molecules eachappear to assist co-stimulation by interacting with their cognateligands on the T cells. Co-stimulatory molecules mediate co-stimulatorysignal(s), which are necessary, under normal physiological conditions,to achieve full activation of naive T cells. One exemplaryreceptor-ligand pair is the B7 co-stimulatory molecule on the surface ofAPCs and its counter-receptor CD28 or CTLA-4 cells (Freeman et al.(1993) Science 262:909-911; Young et al. (1992) J. Clin. Invest. 90:229and Nabavi et al. (1992) Nature 360:266-268). Other importantco-stimulatory molecules are CD40, CD54, CD80, and CD86. The term“co-stimulatory molecule” encompasses any single molecule or combinationof molecules which, when acting together with a peptide/MHC complexbound by a TCR on the surface of a T cell, provides a co-stimulatoryeffect which achieves activation of the T cell that binds the peptide.The term thus encompasses B7, or other co-stimulatory molecule(s) on anantigen-presenting matrix such as an APC, fragments thereof (alone,complexed with another molecule(s), or as part of a fusion protein)which, together with peptide/MHC complex, binds to a cognate ligand andresults in activation of the T cell when the TCR on the surface of the Tcell specifically binds the peptide. Co-stimulatory molecules arecommercially available from a variety of sources, including, forexample, Beckman Coulter, Inc. (Fullerton, Calif.). It is intended,although not always explicitly stated, that molecules having similarbiological activity as wild-type or purified co-stimulatory molecules(e.g., recombinantly produced or muteins thereof) are intended to beused within the spirit and scope of the disclosure.

As used herein, “solid phase support” or “solid support”, usedinterchangeably, is not limited to a specific type of support. Rather alarge number of supports are available and are known to one of ordinaryskill in the art. Solid phase supports include silica gels, resins,derivatized plastic films, glass beads, cotton, plastic beads, aluminagels. As used herein, “solid support” also includes syntheticantigen-presenting matrices, cells, and liposomes. A suitable solidphase support may be selected on the basis of desired end use andsuitability for various protocols. For example, for peptide synthesis,solid phase support may refer to resins such as polystyrene (e.g.,PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.),POLYHIPE® resin (obtained from Aminotech, Canada), polyamide resin(obtained from Peninsula Laboratories), polystyrene resin grafted withpolyethylene glycol (TentaGel®, Rapp Polymere, Tubingen, Germany) orpolydimethylacrylamide resin (obtained from Milligen/Biosearch, Calif.).

An example of a solid phase support include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, gabbros, and magnetite. The natureof the carrier can be either soluble to some extent or insoluble. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding to apolynucleotide, polypeptide or antibody. Thus, the support configurationmay be spherical, as in a bead, or cylindrical, as in the inside surfaceof a test tube, or the external surface of a rod. Alternatively, thesurface may be flat such as a sheet, test strip, etc. or alternativelypolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The term “immunomodulatory agent”, as used herein, is a molecule, amacromolecular complex, or a cell that modulates an immune response andencompasses a synthetic antigenic peptide disclosed herein alone or inany of a variety of formulations described herein; a polypeptidecomprising a synthetic antigenic peptide disclosed herein; apolynucleotide encoding a peptide or polypeptide disclosed herein; asynthetic antigenic peptide disclosed herein bound to a Class I or aClass II MHC molecule on an antigen-presenting matrix, including an APCand a synthetic antigen-presenting matrix (in the presence or absence ofco-stimulatory molecule(s)); a synthetic antigenic peptide disclosedherein covalently or non-covalently complexed to another molecule(s) ormacromolecular structure; and an educated, antigen-specific immuneeffector cell which is specific for a peptide disclosed herein.

The term “modulate an immune response” includes inducing (increasing,eliciting) an immune response; and reducing (suppressing) an immuneresponse. An immunomodulatory method (or protocol) is one that modulatesan immune response in a subject.

As used herein, the term “cytokine” refers to any one of the numerousfactors that exert a variety of effects on cells, for example, inducinggrowth or proliferation. Non-limiting examples of cytokines which may beused alone or in combination in the practice of the present disclosureinclude, interleukin-2 (IL-2), stem cell factor (SCF), interleukin 3(IL-3), interleukin 6 (IL-6), interleukin 12 (IL-12), G-CSF, granulocytemacrophage-colony stimulating factor (GM-CSF), interleukin-1 alpha(IL-1α), interleukin-11 (IL-11), MIP-11, leukemia inhibitory factor(LIF), c-kit ligand, thrombopoietin (TPO) and flt3 ligand. The presentdisclosure also includes culture conditions in which one or morecytokine is specifically excluded from the medium. Cytokines arecommercially available from several vendors such as, for example,Genzyme (Framingham, Mass.), Genentech (South San Francisco, Calif.),Amgen (Thousand Oaks, Calif.), R&D Systems (Minneapolis, Minn.) andImmunex (Seattle, Wash.). It is intended, although not always explicitlystated, that molecules having similar biological activity as wild-typeor purified cytokines (e.g., recombinantly produced or muteins thereof)are intended to be used within the spirit and scope of the disclosure.

Diagnostic and Therapeutic Methods

A method is provided for inhibiting, competing or titrating the bindingof a DNABII polypeptide or protein to a microbial DNA, by contacting theDNABII polypeptide or protein or the microbial DNA with an interferingagent, thereby inhibiting, competing or titrating the binding of theDNABII protein or polypeptide to the microbial DNA. In a further aspect,the DNABII polypeptide and the microbial DNA are detectably labeled, forexample with luminescent molecules that will emit a signal when broughtinto close contact with each other. The contacting can be performed invitro or in vivo.

In another aspect, a method for inhibiting, preventing or breaking downa microbial biofilm is provided by contacting the biofilm with aninterfering agent, thereby inhibiting, preventing or breaking down themicrobial biofilm. In a further aspect, the DNABII polypeptide and themicrobial DNA are detectably labeled, for example with luminescentmolecules that will emit a signal when brought into close contact witheach other. The contacting can be performed in vitro or in vivo.

When practiced in vitro, the methods are useful to screen for or confirminterfering agents having the same, similar or opposite ability as thepolypeptides, polynucleotides, antibodies, host cells, small moleculesand compositions disclosed herein. Alternatively, they can be used toidentify which interfering agent is best suited to treat a microbialinfection or if the treatment has been effective. For example, one canscreen for new agents or combination therapies by having two samplescontaining for example, the DNABII polypeptide and microbial DNA and theagent to be tested. The second sample contains the DNABII polypeptideand microbial DNA and an agent known to active, e.g., an anti-IHFantibody or a small molecule to serve as a positive control. In afurther aspect, several samples are provided and the interfering agentsare added to the system in increasing dilutions to determine the optimaldose that would likely be effective in treating a subject in theclinical setting. As is apparent to those of skill in the art, anegative control containing the DNABII polypeptide and the microbial DNAcan be provided. In a further aspect, the DNABII polypeptide and themicrobial DNA are detectably labeled, for example with luminescentmolecules that will emit a signal when brought into close contact witheach other. The samples are contained under similar conditions for aneffective amount of time for the agent to inhibit, compete or titratethe interaction between the DNABII polypeptide and microbial DNA andthen the sample is assayed for emission of signal from the luminescentmolecules. If the sample emits a signal, then the agent is not effectiveto inhibit binding.

In another aspect, the in vitro method is practiced in a miniaturizedchamber slide system wherein the microbial (such as a bacterial) isolatecausing an infection could be isolated from the human/animal thencultured to allow it to grow as a biofilm in vitro, see for exampleExperiment No. 1 below. The interfering agent (such as anti-DNABII orIHF antibody) or potential interfering agent biofilm is added alone orin combination with another agent to the culture with or withoutincreasing dilutions of the potential interfering agent or interferingagent such as an anti-DNABII or IHF (or other antibody, small molecule,agent, etc.) to find the optimal dose that would likely be effective attreating that patient when delivered to the subject where the infectionexisted. As apparent to those of skill in the art, a positive andnegative control can be performed simultaneously.

In a further aspect, the method is practiced in a high throughputplatform with the interfering agent (such as anti-DNABII or IHFantibody) and/or potential interfering agent (alone or in combinationwith another agent) in a flow cell. The interfering agent (such asanti-DNABII or IHF antibody) or potential interfering agent biofilm isadded alone or in combination with another agent to the culture with orwithout increasing dilutions of the potential interfering agent orinterfering agent such as an anti-DNABII or IHF (or other antibody,small molecule, agent, etc.) to find the optimal dose that would likelybe effective at treating that patient when delivered to the subjectwhere the infection existed. Biofilm isolates are sonicated to separatebiofilm bacteria from DNABII polypeptide such as IHF bound to microbialDNA. The DNABII polypeptide-DNA complexes are isolated by virtue of theanti-DNABII or IHF antibody on the platform. The microbial DNA is thenbe released with e.g., a salt wash, and used to identify the biofilmbacteria added. The freed DNA is then identified, e.g., by PCRsequenced. If DNA is not freed, then the interfering agent(s)successfully performed or bound the microbial DNA. If DNA is found inthe sample, then the agent did not interfere with DNABIIpolypeptide-microbial DNA binding. As is apparent to those of skill inthe art, a positive and/or negative control can be simultaneouslyperformed.

The above methods also can be used as a diagnostic test since it ispossible that a given bacterial species will respond better to reversalof its biofilm by one agent more than another, this rapid highthroughput assay system could allow one skilled the art to assay a panelof possible anti-DNABII or IHF-like agents to identify the mostefficacious of the group.

The advantage of these methods is that most clinical microbiology labsin hospitals are already equipped to perform these sorts of assays(i.e., determination of MIC, MBC values) using bacteria that are growingin liquid culture (or planktonically). As is apparent to those of skillin the art, bacteria generally do not grow planktonic ally when they arecausing diseases. Instead they are growing as a stable biofilm and thesebiofilms are significantly more resistant to treatment by antibiotics,antibodies or other therapeutics. This resistance is why most MIC/MBCvalues fail to accurately predict efficacy in vivo. Thus, by determiningwhat “dose” of agent could reverse a bacterial biofilm in vitro (asdescribed above) Applicants' pre-clinical assay would be a more reliablepredictor of clinical efficacy, even as an application of personalizedmedicine.

In addition to the clinical setting, the methods can be used to identifythe microbe causing the infection and/or confirm effective interferingagents in an industrial setting. Thus, the interfering agents can beused to treat, inhibit or titrate a biofilm in an industrial setting.

In a further aspect of the above methods, an antibiotic or antimicrobialknown to inhibit growth of the underlying infection is addedsequentially or concurrently, to determine if the infection can beinhibited. It is also possible to add the interfering agent to themicrobial DNA or DNABII polypeptide before adding the missing complex toassay for biofilm inhibition.

When practiced in vivo in non-human animal such as a chinchilla, themethod provides a pre-clinical screen to identify interfering agentsthat can be used alone or in combination with other agents to break downbiofilms. Examples of this method are shown in Experiment Nos. 2 through7, below.

In another aspect, provided herein is a method of inhibiting, preventingor breaking down a biofilm in a subject by administering to the subjectan effective amount of an interfering agent, thereby inhibiting,preventing or breaking down the microbial biofilm. Examples of thismethod are shown in Experiment Nos. 2 through 7, below. Non-limitingexamples of such subjects include mammals, e.g., pets, and humanpatients.

For the purpose of the above noted in vitro and in vivo methods, theinterfering agent is of the group of:

(a) an isolated or recombinant DNABII or an integration host factor(IHF) polypeptide or a fragment or an equivalent of each thereof;

(b) an isolated or recombinant histone-like protein from E. coli, e.g.,E. coli strain U93 (HU) polypeptide or a fragment or an equivalent ofeach thereof;

(c) an isolated or recombinant protein or polypeptide identified inTable 8, FIG. 19 , FIG. 20 , Table 9 or a DNA binding peptide identifiedin FIGS. 6A-6B, or a fragment or an equivalent of each thereof, whereinin one aspect the fragment comprises, or consists essentially of, or yetconsists of, the polypeptides identified as A1 through A6 or B1 throughB6 as disclosed in FIG. 18 ; or comprising, or alternatively consistingessentially of, or yet further consisting of the “tip” portion of theDNABII protein or polypeptide, comprising, or alternatively consistingessentially of, or yet further consisting of KLSGFGNFELRDKSSRPGRN (alsoreferred to herein as hIFA4; (SEQ ID NO. 351); RPGRNPKTGDVVPVSARRVV(also referred to herein as hIFA5; (SEQ ID NO. 352);ARRVVTFKPGQKLRARVEKTK (also referred to herein as hIFA6; (SEQ ID NO.353), or an equivalent of each thereof, or an equivalent of each thereofor a polypeptide having at least 60%, or alternatively at least 65%, oralternatively at least 70%, or alternatively at least 75%, oralternatively 80%, or alternatively at least 85%, or alternatively atleast 90%, or alternatively at least 95% identity thereto or forpolypeptide sequences, which is encoded by a polynucleotide or itscomplement that hybridizes under conditions of high stringency to apolynucleotide encoding such polypeptide sequences. Conditions of highstringency are described herein and incorporated herein by reference.Applicants have determined that the bolded and underlined amino acidsare heavily conserved and therefore in one aspect, are not modified oraltered in designing an equivalent polypeptide. Additional examples ofequivalent polypeptides include, for example IEYLSDKYHLSKQDTK (SEQ IDNO. 354), DKSSRPGRNPKTGDVVAASARR (SEQ ID NO.: 355), and KLRARVEKTK (SEQID NO. 17), described in U.S. Ser. Nos. 14/497,147 and 14/668,767 nowabandoned). Equivalent polypeptides also include a polypeptideconsisting of or comprising the above noted polypeptides with theaddition of up to 25, or alternatively 20, or alternatively 15, oralternatively up to 10, or alternatively up to 5 random amino acids oneither the amine or carboxy termini (or on both);(d) an isolated or recombinant polypeptide of SEQ ID NO: 1 through 353,or a fragment or an equivalent of each thereof;(e) an isolated or recombinant C-terminal polypeptide of SEQ ID NO: 6through 11, 28, 29, 42 through 100, Table 8 or those C-terminalpolypeptides identified in Table 9 or a fragment or an equivalent ofeach thereof;(f) a polypeptide or polynucleotide that competes with a DNABII proteinon binding to a microbial DNA, e.g., DNABII or an integration hostfactor IHF and/or HU polypeptide or protein;(g) a four-way junction polynucleotide resembling a Holliday junction, a3 way junction polynucleotide resembling a replication fork, apolynucleotide that has inherent flexibility or bent polynucleotide;(h) an isolated or recombinant polynucleotide encoding any one of (a)through (f) or an isolated or recombinant polynucleotide of SEQ ID NO:36 or an equivalent of each thereof, or a polynucleotide that hybridizesunder stringent conditions to the polynucleotide its equivalent or itscomplement;(i) an antibody or antigen binding fragment that specifically recognizesor binds any one of (a) through (g), or an equivalent or fragment ofeach antibody or antigen binding fragment thereof;(j) an isolated or recombinant polynucleotide encoding the antibody orantigen binding fragment of (i) or its complement;(k) a small molecule that competes with the binding of a DNABII proteinor polypeptide to a microbial DNA;(l) an antibody or antigen binding fragment that specifically recognizesor binds any one of an isolated or recombinant polypeptide of SEQ ID NO:342 through 353 or a fragment or an equivalent of each thereof; and/or(m) polypeptide that comprises, or alternatively consists essentiallyof, or yet further consists of polypeptides A1 through A6 or B1 throughB6 as disclosed in FIG. 18 ; or KLSGFGNFELRDKSSRPGRN (also referred toherein as hIFA4; (SEQ ID NO. 351); RPGRNPKTGDVVPVSARRVV (also referredto herein as hIFA5; (SEQ ID NO. 352); ARRVVTFKPGQKLRARVEKTK (alsoreferred to herein as hIFA6; (SEQ ID NO. 353), or an equivalent of eachthereof or a polypeptide having at least 60%, or alternatively at least65%, or alternatively at least 70%, or alternatively at least 75%, oralternatively 80%, or alternatively at least 85%, or alternatively atleast 90%, or alternatively at least 95% identity thereto or forpolypeptide sequences, which is encoded by a polynucleotide or itscomplement that hybridizes under conditions of high stringency to apolynucleotide encoding such polypeptide sequences. Conditions of highstringency are described herein and incorporated herein by reference.Applicants have determined that the bolded and underlined amino acidsare heavily conserved and therefore in one aspect, are not modified oraltered in designing an equivalent polypeptide. Additional examples ofequivalent polypeptides include, for example IEYLSDKYHLSKQDTK (SEQ IDNO. 354), DKSSRPGRNPKTGDVVAASARR (SEQ ID NO.: 355), and KLRARVEKTK (SEQID NO. 17) described in U.S. Ser. Nos. 14/497,147 and 14/668,767 nowabandoned), a polypeptide consisting of or comprising the above notedpolypeptides with the addition of up to 25, or alternatively 20, oralternatively 15, or alternatively up to 10, or alternatively up to 5random amino acids on either the amine or carboxy termini (or on both).

Also provided herein is a method for inducing an immune response in orconferring passive immunity in a subject in need thereof, comprising, oralternatively consisting essentially of or yet further consisting of,administering to the subject an effective amount of one or more agentsof the group:

(a) an isolated or recombinant DNABII or an integration host factor(INF) polypeptide, or a fragment or an equivalent of each thereof;

(b) an isolated or recombinant histone-like protein from E. coli, e.g.,E. coli strain U93 (HU) polypeptide or a fragment or an equivalent ofeach thereof;

(c) an isolated or recombinant protein polypeptide identified in Table8, FIG. 19 , FIG. 20 , Table 9 or an DNA binding peptide identified inFIGS. 6A-6B, or a fragment or an equivalent of each thereof, apolypeptide A1 to A5, or B1 to B5 as shown in FIG. 18 , or a fragmentthat comprises, or consists essentially of, or yet consists of, the“tip” portion of the DNABII protein or polypeptide, non-limitingexamples of such include without limitation a polypeptide thatcomprises, or alternatively consists essentially of, or yet furtherconsists of KLSGFGNFELRDKSSRPGRN (also referred to herein as hIFA4; (SEQID NO. 351); RPGRNPKTGDVVPVSARRVV (also referred to herein as hIFA5;(SEQ ID NO. 352); ARRVVTFKPGQKLRARVEKTK (also referred to herein ashIFA6; (SEQ ID NO. 353), or an equivalent of each thereof. Additionalexamples of equivalent polypeptides include, for exampleIEYLSDKYHLSKQDTK (SEQ ID NO. 354), DKSSRPGRNPKTGDVVAASARR (SEQ ID NO.:355), AND KLRARVEKTK (SEQ ID NO. 17) described in U.S. Ser. Nos.14/497,147 and 14/668,767 now abandoned), a polypeptide consisting of orcomprising the above noted polypeptides with the addition of up to 25,or alternatively 20, or alternatively 15, or alternatively up to 10, oralternatively up to 5 random or naturally occurring amino acids oneither the amine or carboxy termini (or on both);(d) an isolated or recombinant polypeptide of SEQ ID NO: 1 through 353or a fragment or an equivalent thereof;(e) an isolated or recombinant C-terminal polypeptide of SEQ ID NO: 6through 11, 28, 29, 42 through 100, Table 8 or those C-terminalpolypeptides identified in Table 9 or a fragment or an equivalent ofeach thereof;(f) an isolated or recombinant polynucleotide encoding any one of (a)through (e) or an isolated or recombinant polynucleotide SEQ JD NO: 36or an equivalent of each thereof, or a polynucleotide that hybridizesunder stringent conditions to the polynucleotide, its equivalent or itscomplement;(g) an antibody or antigen binding fragment that specifically recognizesor binds any one of (a) through (e), or an equivalent or fragment ofeach thereof;(h) an isolated or recombinant polynucleotide encoding the antibody orantigen binding fragment of (g);(i) an antigen presenting cell pulsed with any one of (a) through (e);(j) an antigen presenting cell transfected with one or morepolynucleotides encoding any one of (a) through (e); and/or(k) an antibody or antigen binding fragment that specifically recognizesor binds any one of an isolated or recombinant polypeptide noted hereinas A1, A2, A3, A4, A5, A6, B1, B2, B3, B4, B5, B6 (se FIG. 18 ) or anisolated or recombinant polypeptide of SEQ ID NO: 342 through 353 or afragment or an equivalent of each thereof.

In one particular aspect, the interfering agent is an isolated orrecombinant DNABII protein, e.g., an integration host factor (IHF)polypeptide or a fragment thereof, a C-terminal fragment of an IHFpolypeptide, a tip fragment, of an equivalent of each thereof. Inanother particular aspect, the interfering agent is an isolated orrecombinant HU polypeptide or a fragment thereof, a tip fragment, aC-terminal fragment of HU polypeptide, or an equivalent of each thereof.Non-limiting examples of such are an IHF or HU alpha or betapolypeptide; an IHF polypeptide; Moraxella catarrhalis HU; E. coli HupA,HupB, himA, himD; E. faecalis HU (such as V583), HMGB1 (High MobilityGroup Box 1, a protein with similar DNA binding and DNA substratespecificities but not in primary amino acid sequence to the DNABIIfamily of proteins; a functional orthologue) and those identified inTable 8 or Table 9.

In a further aspect, the methods further comprise, or alternativelyconsist essentially of, or yet further consist of administering to thesubject an effective amount of one or more of an antimicrobial, anantigenic peptide or an adjuvant.

A non-limiting example of an antimicrobial agent is another vaccinecomponent such as a surface antigen, e.g., an OMP P5, rsPilA, OMP 26,OMP P2, or Type IV Pilin protein (see Jurcisek and Bakaletz (2007) J.Bacteriology 189(10):3868-3875; Murphy, T. F. et al. (2009) ThePediatric Infectious Disease Journal 28:S121-S126).

The agents and compositions disclosed herein can be concurrently orsequentially administered with other antimicrobial agents and/or surfaceantigens. In one particular aspect, administration is locally to thesite of the infection by direct injection or by inhalation for example.Other non-limiting examples of administration include by one or moremethod comprising transdermally, urethrally, sublingually, rectally,vaginally, ocularly, subcutaneous, intramuscularly, intraperitoneally,intranasally, by inhalation or orally.

Microbial infections and disease that can be treated by the methodsdisclosed herein include infection by the organisms identified inExperiment No. 1 and Table 8, e.g., Streptococcus agalactiae, Neisseriameningitidis, Treponemes, denticola, pallidum, Burkholderia cepacia, orBurkholderia pseudomallei. In one aspect, the microbial infection is oneor more of Haemophilus influenzae (nontypeable), Moraxella catarrhalis,Streptococcus pneumoniae, Streptococcus pyogenes, Pseudomonasaeruginosa, Mycobacterium tuberculosis. These microbial infections maybe present in the upper, mid and lower airway (otitis, sinusitis,bronchitis but also exacerbations of chronic obstructive pulmonarydisease (COPD), chronic cough, complications of and/or primary cause ofcystic fibrosis (CF) and community acquired pneumonia (CAP). Thus, bypracticing the in vivo methods disclosed herein, these diseases andcomplications from these infections can also be prevented or treated.

Infections might also occur in the oral cavity (caries, periodontitis)and caused by Streptococcus mutans, Porphyromonas gingivalis,Aggregatibacter actinomycetemcomitans. Infections might also belocalized to the skin (abscesses, ‘staph’ infections, impetigo,secondary infection of burns, Lyme disease) and caused by Staphylococcusaureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Borreliaburdorferi. Infections of the urinary tract (UTI) can also be treatedand are typically caused by Escherichia coli. Infections of thegastrointestinal tract (GI) (diarrhea, cholera, gall stones, gastriculcers) are typically caused by Salmonella enterica serovar, Vibriocholerae and Helicobacter pylori. Infections of the genital tractinclude and are typically caused by Neisseria gonorrhoeae. Infectionscan be of the bladder or of an indwelling device caused by Enterococcusfaecalis. Infections associated with implanted prosthetic devices, suchas artificial hip or knee replacements, or dental implants, or medicaldevices such as pumps, catheters, stents, or monitoring systems,typically caused by a variety of bacteria, can be treated by the methodsdisclosed herein. These devices can be coated or conjugated to an agentas described herein. Thus, by practicing the in vivo methods disclosedherein, these diseases and complications from these infections can alsobe prevented or treated.

Infections caused by Streptococcus agalactiae can also be treated by themethods disclosed herein and it is the major cause of bacterialsepticemia in newborns. Infections caused by Neisseria meningitidiswhich can cause meningitis can also be treated.

Thus, routes of administration applicable to the methods disclosedherein include intranasal, intramuscular, urethrally, intratracheal,subcutaneous, intradermal, transdermal, topical application,intravenous, rectal, nasal, oral, inhalation, and other enteral andparenteral routes of administration. Routes of administration may becombined, if desired, or adjusted depending upon the agent and/or thedesired effect. An active agent can be administered in a single dose orin multiple doses. Embodiments of these methods and routes suitable fordelivery include systemic or localized routes. In general, routes ofadministration suitable for the methods disclosed herein include, butare not limited to, direct injection, enteral, parenteral, orinhalational routes.

Parenteral routes of administration other than inhalation administrationinclude, but are not limited to, topical, transdermal, subcutaneous,intramuscular, intraorbital, intracapsular, intraspinal, intrasternal,and intravenous routes, i.e., any route of administration other thanthrough the alimentary canal. Parenteral administration can be conductedto effect systemic or local delivery of the inhibiting agent. Wheresystemic delivery is desired, administration typically involves invasiveor systemically absorbed topical or mucosal administration ofpharmaceutical preparations.

The interfering agents disclosed herein can also be delivered to thesubject by enteral administration. Enteral routes of administrationinclude, but are not limited to, oral and rectal (e.g., using asuppository) delivery.

Methods of administration of the active through the skin or mucosainclude, but are not limited to, topical application of a suitablepharmaceutical preparation, transcutaneous transmission, transdermaltransmission, injection and epidermal administration. For transdermaltransmission, absorption promoters or iontophoresis are suitablemethods. Iontophoretic transmission may be accomplished usingcommercially available “patches” that deliver their product continuouslyvia electric pulses through unbroken skin for periods of several days ormore.

In various embodiments of the methods disclosed herein, the interferingagent will be administered by inhalation, injection or orally on acontinuous, daily basis, at least once per day (QD), and in variousembodiments two (BID), three (TID), or even four times a day. Typically,the therapeutically effective daily dose will be at least about 1 mg, orat least about 10 mg, or at least about 100 mg, or about 200 to about500 mg, and sometimes, depending on the compound, up to as much as about1 g to about 2.5 g.

Dosing of can be accomplished in accordance with the methods disclosedherein using capsules, tablets, oral suspension, suspension forintra-muscular injection, suspension for intravenous infusion, get orcream for topical application, or suspension for intra-articularinjection.

Dosage, toxicity and therapeutic efficacy of compositions describedherein can be determined by standard pharmaceutical procedures in cellcultures or experimental animals, for example, to determine the LD50(the dose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD50/ED50. In certain embodiments,compositions exhibit high therapeutic indices. While compounds thatexhibit toxic side effects may be used, care should be taken to design adelivery system that targets such compounds to the site of affectedtissue in order to minimize potential damage to uninfected cells and,thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies (in certain embodiments, within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods, the therapeutically effective dose can beestimated initially from cell culture assays. A dose can be formulatedin animal models to achieve a circulating plasma concentration rangethat includes the IC50 (i.e., the concentration of the test compoundwhich achieves a half-maximal inhibition of symptoms) as determined incell culture. Such information can be used to more accurately determineuseful doses in humans. Levels in plasma may be measured, for example,by high performance liquid chromatography.

In some embodiments, an effective amount of a composition sufficient forachieving a therapeutic or prophylactic effect, ranges from about0.000001 mg per kilogram body weight per administration to about 10,000mg per kilogram body weight per administration. Suitably, the dosageranges are from about 0.0001 mg per kilogram body weight peradministration to about 100 mg per kilogram body weight peradministration. Administration can be provided as an initial dose,followed by one or more “booster” doses. Booster doses can be provided aday, two days, three days, a week, two weeks, three weeks, one, two,three, six or twelve months after an initial dose. In some embodiments,a booster dose is administered after an evaluation of the subject'sresponse to prior administrations.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to, the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of the therapeutic compositionsdescribed herein can include a single treatment or a series oftreatments.

Polypeptides

Also provided herein are interfering agents and compositions for use inthe methods described herein, wherein the interfering agent is of thegroup:

(a) an isolated or recombinant DNABII or an integration host factor(IHF) polypeptide or a fragment or an equivalent of each thereof;

(b) an isolated or recombinant histone-like protein from E. coli, e.g.,E. coli strain U93 (HU) polypeptide or a fragment or an equivalent ofeach thereof;

(c) an isolated or recombinant protein or polypeptide identified inTable 8, FIG. 19 , FIG. 20 , Table 9 or a DNA binding peptide identifiedin FIGS. 6A-6B, or a fragment or an equivalent of each thereof, whereinin one aspect the fragment comprises, or consists essentially of, or yetconsists of, the polypeptides identified as A1 through A6 or B1 throughB6 as disclosed in FIG. 18 ; or comprising, or alternatively consistingessentially of, or yet further consisting of the “tip” portion of theDNABII protein or polypeptide, comprising, or alternatively consistingessentially of, or yet further consisting of KLSGFGNFELRDKSSRPGRN (alsoreferred to herein as hIFA4; (SEQ ID NO. 351); RPGRNPKTGDVVPVSARRVV(also referred to herein as hIFA5; (SEQ ID NO. 352);ARRVVTFKPGQKLRARVEKTK (also referred to herein as hIFA6; (SEQ ID NO.353); or an equivalent of each thereof; or an equivalent of each thereofor a polypeptide having at least 60%, or alternatively at least 65%, oralternatively at least 70%, or alternatively at least 75%, oralternatively 80%, or alternatively at least 85%, or alternatively atleast 90%, or alternatively at least 95% identity thereto or forpolypeptide sequences, which is encoded by a polynucleotide or itscomplement that hybridizes under conditions of high stringency to apolynucleotide encoding such polypeptide sequences. Conditions of highstringency are described herein and incorporated herein by reference.Applicants have determined that the bolded and underlined amino acidsare heavily conserved and therefore in one aspect, are not modified oraltered in designing an equivalent polypeptide. Additional examples ofequivalent polypeptides include, for example IEYLSDKYHLSKQDTK (SEQ IDNO. 354), DKSSRPGRNPKTGDVVAASARR (SEQ ID NO.: 355), and KLRARVEKTK (SEQID NO. 17), described in U.S. Ser. Nos. 14/497,147 and 14/668,767 nowabandoned). Equivalent polypeptides also include a polypeptideconsisting of or comprising the above noted polypeptides with theaddition of up to 25, or alternatively 20, or alternatively 15, oralternatively up to 10, or alternatively up to 5 random amino acids oneither the amine or carboxy termini (or on both);(d) an isolated or recombinant polypeptide of SEQ ID NO: 1 through 353,or a fragment or an equivalent of each thereof;(e) an isolated or recombinant C-terminal polypeptide of SEQ ID NO: 6through 11, 28, 29, 42 through 100, Table 8 or those C-terminalpolypeptides identified in Table 9 or a fragment or an equivalent ofeach thereof;(f) a polypeptide or polynucleotide that competes with a DNABII proteinon binding to a microbial DNA, e.g., DNABII or an integration hostfactor IHF and/or HU polypeptide or protein;(g) a four-way junction polynucleotide resembling a Holliday junction, a3 way junction polynucleotide resembling a replication fork, apolynucleotide that has inherent flexibility or bent polynucleotide;(h) an isolated or recombinant polynucleotide encoding any one of (a)through (f) or an isolated or recombinant polynucleotide of SEQ ID NO:36 or an equivalent of each thereof, or a polynucleotide that hybridizesunder stringent conditions to the polynucleotide its equivalent or itscomplement;(i) an antibody or antigen binding fragment that specifically recognizesor binds any one of (a) through (g), or an equivalent or fragment ofeach antibody or antigen binding fragment thereof;(j) an isolated or recombinant polynucleotide encoding the antibody orantigen binding fragment of (i) or its complement;(k) a small molecule that competes with the binding of a DNABII proteinor polypeptide to a microbial DNA;(l) an antibody or antigen binding fragment that specifically recognizesor binds any one of an isolated or recombinant polypeptide of SEQ ID NO:342 through 353 or a fragment or an equivalent of each thereof; and/or(m) polypeptide that comprises, or alternatively consists essentiallyof, or yet further consists of polypeptides A1 through A6 or B1 throughB6, as disclosed in FIG. 18 ; or KLSGFGNFELRDKSSRPGRN (also referred toherein as hIFA4; (SEQ ID NO. 351); RPGRNPKTGDVVPVSARRVV (also referredto herein as hIFA5; (SEQ ID NO. 352); ARRVVTFKPGQKLRARVEKTK (alsoreferred to herein as hIFA6; (SEQ ID NO. 353), or an equivalent of eachthereof or a polypeptide having at least 60%, or alternatively at least65%, or alternatively at least 70%, or alternatively at least 75%, oralternatively 80%, or alternatively at least 85%, or alternatively atleast 90%, or alternatively at least 95% identity thereto or forpolypeptide sequences, which is encoded by a polynucleotide or itscomplement that hybridizes under conditions of high stringency to apolynucleotide encoding such polypeptide sequences. Conditions of highstringency are described herein and incorporated herein by reference.Applicants have determined that the bolded and underlined amino acidsare heavily conserved and therefore in one aspect, are not modified oraltered in designing an equivalent polypeptide. Additional examples ofequivalent polypeptides include, for example IEYLSDKYHLSKQDTK (SEQ IDNO. 354), DKSSRPGRNPKTGDVVAASARR (SEQ ID NO.: 355), and KLRARVEKTK (SEQID NO. 17) described in U.S. Ser. Nos. 14/497,147 and 14/668,767 nowabandoned), a polypeptide consisting of or comprising the above notedpolypeptides with the addition of up to 25, or alternatively 20, oralternatively 15, or alternatively up to 10, or alternatively up to 5random amino acids on either the amine or carboxy termini (or on both).

In one particular aspect, the interfering agent is an isolated orrecombinant DNABII polypeptide or a fragment or an equivalent of eachthereof. Non-limiting examples of such are an IHF or HU alpha or betapolypeptide; an IHF a polypeptide; Moraxella catarrhalis Flu; E. coliHupA, HupB, himA, himD; E. faecalis HU (such as V583), HMGB1 and thoseidentified in Table 8.

In another aspect, the interfering agent is an isolated or recombinantpolypeptide consisting essentially of an amino acid sequence selectedfrom SEQ ID NO: 1 to 5 or 12 to 27, 30 to 35, 101-353 or a DNA bindingpeptide identified in FIGS. 6A-6B.

In another aspect, the isolated or recombinant polypeptide comprises, oralternatively consists essentially of or yet further consists of SEQ IDNO: 1 or 2, with the proviso that the polypeptide is none of SEQ ID NO:6 to 11, 28, 29, or 42 through 100.

In another aspect, the isolated or recombinant polypeptide comprises, oralternatively consists essentially of, or yet further consists of SEQ IDNO: 3, 4 or 5, with the proviso that the polypeptide is none of SEQ IDNO: 6 to 11, 28, 29, or 42 through 100.

In another aspect, the isolated or recombinant polypeptide comprises, oralternatively consists essentially of, or yet further consists of SEQ IDNO: 12, 14, 16, 18, 20, 22, 24, 26, 30 or 32, with the proviso that thepolypeptide is none of SEQ ID NO: 6 to 11, 28, 29, or 42 through 100.

In another aspect, the isolated or recombinant polypeptide comprises, oralternatively consists essentially of, or yet further consists of SEQ IDNO: 13, 15, 17, 19, 21, 23, 25, 27, 31 33, 34, or 35 with the provisothat the polypeptide is none of SEQ ID NO: 6 to 11, 28, 29, or 42through 100.

In another aspect, the isolated or recombinant polypeptide comprises, oralternatively consists essentially of, or yet further consists of anisolated or recombinant polypeptide of the group of:

a polypeptide comprising SEQ ID NO: 12 and 13;

a polypeptide comprising SEQ ID NO: 14 and 15;

a polypeptide comprising SEQ ID NO: 16 and 17;

a polypeptide comprising SEQ ID NO: 18 and 19;

a polypeptide comprising SEQ ID NO: 20 and 21;

a polypeptide comprising SEQ ID NO: 23 and 24;

a polypeptide comprising SEQ ID NO: 25 and 26;

a polypeptide comprising SEQ ID NO: 30 and 31;

a polypeptide comprising SEQ ID NO: 32 and 33;

a polypeptide comprising SEQ ID NO: 34 and 35;

a polypeptide comprising SEQ ID NO: 337 and 338; or

a polypeptide comprising SEQ ID NO: 339 and 340;

a polypeptide consisting essentially of any one or more of SEQ ID NO:342 to 353;

with the proviso that the polypeptide is none of wild-type of any one ofIHF alpha, IHF beta or SEQ ID NO: 6 to 11, 28, 29, or 42 through 100.

In another aspect, the isolated or recombinant polypeptide is of thegroup:

a polypeptide consisting essentially of SEQ ID NO: 12 and 13;

a polypeptide consisting essentially of SEQ ID NO: 14 and 15;

a polypeptide consisting essentially of SEQ ID NO: 16 and 17;

a polypeptide consisting essentially of SEQ ID NO: 18 and 19;

a polypeptide consisting essentially of SEQ ID NO: 20 and 21;

a polypeptide consisting essentially of SEQ ID NO: 23 and 24;

a polypeptide consisting essentially of SEQ ID NO: 25 and 26;

a polypeptide consisting essentially of SEQ ID NO: 30 and 31;

a polypeptide consisting essentially of SEQ ID NO: 32 and 33;

a polypeptide consisting essentially of SEQ ID NO: 34 and 35;

a polypeptide consisting essentially of SEQ ID NO: 337 and 338; or

a polypeptide consisting essentially of SEQ ID NO: 339 and 340;

a polypeptide consisting essentially of any one or more of SEQ ID NO:342 to 353;

with the proviso that the polypeptide is none of wild-type of any one ofIHF alpha, IHF beta or SEQ ID NO: 6 to 11, 28, 29, or 42 through 100.

Further provided as agents for use in the methods disclosed herein arefragments or an equivalent of the isolated or recombinant polypeptidesdescribed above. Examples of fragments are A1 through A6; B1 through B6(see FIG. 18 ); or a C-terminal polypeptide. In another aspect, thefragment is a tip portion of the DNABII, non-limiting examples of suchincludes without limitation a polypeptide that comprisesKLSGFGNFELRDKSSRPGRN (also referred to herein as hIFA4; (SEQ ID NO.351)); RPGRNPKTGDVVPVSARRVV (also referred to herein as hIFA5; (SEQ IDNO. 352)); ARRVVTFKPGQKLRARVEKTK (also referred to herein as hIFA6; (SEQID NO. 353)); or an equivalent of each thereof. In a further aspect, theisolated or recombinant polypeptide comprises, or alternatively consistsessentially of, or yet further consists of two or more of the isolatedor recombinant polypeptides described above.

Additionally, the isolated or recombinant polypeptide comprises, oralternatively consists essentially of, or yet further consists of anyone of SEQ ID NO: 1 to 6, 12 to 27 or 30 to 33, or a fragment or anequivalent polypeptide, examples of which are identified in Table 8 orshown in FIG. 19 , FIG. 20 or Table 9. In one aspect, isolated wild-typepolypeptides are excluded, i.e., that the polypeptide is none of SEQ IDNO: 6 through 11, 28, 29, or a wildtype sequence identified in Table 8or shown in FIG. 19 .

In one aspect, this disclosure provides an isolated or recombinantpolypeptide consisting essentially of an amino acid sequence of thegroup SEQ ID NO: 1 to 6, 12 to 27 or 30 to 35, 1 to 6 and 13 to 35, or apolypeptide comprising, or alternatively consisting essentially of, oryet further consisting of an amino acid corresponding to the β-3 and/orα-3 fragments of a Haemophilus influenzae IHFα or IHFβ, non-limitingexamples of which include SEQ ID NO: 12 through 27, or a fragment orequivalent thereof of each thereof. In another aspect, the disclosureprovides an isolated or recombinant polypeptide comprising, oralternatively consisting essentially of, or yet further consisting of anamino acid sequence of the group SEQ ID NO: 1 to 4, or a fragment or anequivalent of each thereof, or a polypeptide comprising, oralternatively consisting essentially of, or yet further consisting of anamino acid corresponding to the β-3 and/or α-3 fragments of aHaemophilus influenzae IHFα or IHFβ, non-limiting examples of whichinclude SEQ ID NO: 12 through 27 or a fragment or a biologicalequivalent thereof which further comprises independently at least 2, oralternatively at least 3, or alternatively at least 4, or alternativelyat least 5, or at least 6, or alternatively at least 7, or alternativelyat least 8, or alternatively at least 9 or alternatively at least 10amino acids at the amino and/or carboxyl terminus of the polypeptide. Inone aspect, isolated wildtype DNA binding polypeptides are excluded,i.e., that the polypeptide is none of SEQ ID NO: 6 through 11, 28, 29,or 42 through 100 or an isolated wildtype polypeptide sequence listed inTable 8 or shown in FIG. 19 .

In another aspect, this disclosure provides an isolated or recombinantpolypeptide comprising, or alternatively consisting essentially of, oryet further consisting of, SEQ ID. NO 1 or 2 alone or in combinationwith a polypeptide comprising, or alternatively consisting essentiallyof, or yet further consisting of an amino acid corresponding to the β-3and/or α-3 fragments of a Haemophilus influenzae IHFα or IHFβ,on-limiting examples of which include SEQ ID NO: 12 through 27 or afragment or a biological equivalent of each thereof. In one aspect,isolated wildtype DNA binding polypeptides are excluded, i.e., that thepolypeptide is none of SEQ ID NO: 6 through 11, 28, 29, or 42 through100 or an isolated polypeptide sequence listed in Table 8 or shown inFIG. 19 .

In a yet further aspect, this disclosure provides an isolated orrecombinant polypeptide comprising or alternatively consistingessentially of, or yet further consisting of, SEQ ID NO: 3 or 4 or afragment or an equivalent of each thereof alone or in combination with apolypeptide comprising, or alternatively consisting essentially of, oryet further consisting of an amino acid corresponding to the β-3 and/orα-3 fragments of a Haemophilus influenzae IHFα or IHFβ, non-limitingexamples of which include SEQ ID NO: 12 through 27, and 34-35 or abiological equivalent of each thereof. In one aspect, isolated wildtypeDNA binding polypeptides are excluded, i.e., that the polypeptide isnone of SEQ ID NO: 6 through 11, 28, 29, or 42 through 100 or anisolated wildtype polypeptide sequence listed in Table 8 or shown inFIG. 19 , or a polypeptide that comprises KLSGFGNFELRDKSSRPGRN (alsoreferred to herein as hIFA4; (SEQ ID NO. 351)); RPGRNPKTGDVVPVSARRVV(also referred to herein as hIFA5; (SEQ ID NO. 352));ARRVVTFKPGQKLRARVEKTK (also referred to herein as hIFA6; (SEQ ID NO.353)), or an equivalent of each thereof.

This disclosure also provides isolated or recombinant polypeptidescomprising or alternatively consisting essentially of, or yet furtherconsisting of, two or more, or three or more, four or more, five ormore, six or more, seven or more, eight or more, nine or more, ten ormore, eleven or more, twelve or more, thirteen or more of all fourteenof the isolated polypeptides or a fragment or an equivalent of eachthereof. Non-limiting examples of such include isolated or recombinantpolypeptides comprising SEQ ID NO: 1 through 4, e.g., SEQ ID NO: 1 and2, or alternatively 1 and 3 or alternatively 1 and 4, or alternatively 2and 3, or alternatively SEQ ID NO: 1, 2 and 3 or alternatively, 2, 3 and4, or alternatively 1, 3 and 4. The polypeptides can be in anyorientation, e.g., SEQ ID NO: 1, 2, and 3 or SEQ ID NO: 3, 2 and 1 oralternatively 2, 1 and 3, or alternatively, 3, 1 and 2. Biologicalequivalents of these polypeptides are further included in thisdisclosure with the proviso that the sequences do not include isolatedwildtype sequences such as those identified in Tables 8 and 9.

In another aspect, this disclosure provides an isolated or recombinantpolypeptide comprising or alternatively consisting essentially of, oryet further consisting of, SEQ ID NO: 1 or 2 and 3 or 4, or a fragmentor an equivalent of each thereof, with the proviso that the polypeptideis none of SEQ ID NO: 5 through 10, and they may further comprise anyone or more of SEQ ID NO: 11 through 26, e.g., 11 and 12, oralternatively 1 and 11, or alternatively 2 and 11, or alternatively, 1and 12, or alternatively 2 and 12, or alternatively 11, 12 and 1, oralternatively 2, 11 and 12. In this embodiment, SEQ ID NO: 1 or 2 islocated upstream or amino terminus from SEQ ID NO: 3 or 4, with theproviso that the amino acid sequence is not an isolated wildtypepolypeptide, e.g., none of SEQ ID NO: 6 through 11, 28 and 29. Inanother aspect, the isolated polypeptide comprises SEQ ID NO: 3 or 4located upstream or amino terminus to SEQ ID NO: 1 or 2. Biologicalequivalents of these polypeptides are further included in thisdisclosure with the proviso that the sequence do not include isolatedwildtype polypeptides.

In one embodiment, any polypeptide or protein having sequence identityto the wildtype polypeptides or those disclosed in Pedulla et al. (1996)PNAS 93:15411-15416 is excluded from this disclosure.

In any of the above embodiments, a peptide linker can be added to theN-terminus or C-terminus of the polypeptide. A “linker” or “peptidelinker” refers to a peptide sequence linked to either the N-terminus orthe C-terminus of a polypeptide sequence. In one aspect, the linker isfrom about 1 to about 20 amino acid residues long or alternatively 2 toabout 10, about 3 to about 5 amino acid residues long. An example of apeptide linker is Gly-Pro-Ser-Leu-Lys-Leu (SEQ ID NO: 37). Otherexamples include Gly-Gly-Gly; Gly-Pro-Ser-Leu (SEQ ID NO: 38);Gly-Pro-Ser; Pro-Ser-Leu-Lys (SEQ ID NO: 39); Gly-Pro-Ser-Leu-Lys (SEQID NO: 40) and Ser-Leu-Lys-Leu (SEQ ID NO: 41).

The isolated polypeptides disclosed herein are intended to includeisolated wildtype and recombinantly produced polypeptides and proteinsfrom prokaryotic and eukaryotic host cells, as well as muteins, analogsand fragments thereof, examples of such cells are described above. Insome embodiments, the term also includes antibodies and anti-idiotypicantibodies as described herein. Such polypeptides can be isolated orproduced using the methods known in the art and briefly describedherein.

It is understood that functional equivalents or variants of the wildtype polypeptide or protein also are within the scope of thisdisclosure, for example, those having conservative amino acidsubstitutions of the amino acids, see for example, Table 9. Otheranalogs include fusion proteins comprising a protein or polypeptidedisclosed herein which can include a polypeptide joined to an antigenpresenting matrix.

In a further aspect, the polypeptides are conjugated or linked to adetectable label. Suitable labels are known in the art and describedherein.

In a yet further aspect, the polypeptides with or without a detectablelabel can be contained or expressed on the surface of a host prokaryoticor eukaryotic host cell, such as a dendritic cell.

The proteins and polypeptides are obtainable by a number of processesknown to those of skill in the art, which include purification, chemicalsynthesis and recombinant methods. Polypeptides can be isolated frompreparations such as host cell systems by methods such asimmunoprecipitation with antibody, and standard techniques such as gelfiltration, ion-exchange, reversed-phase, and affinity chromatography.For such methodology, see for example Deutscher et al. (1999) Guide ToProtein Purification: Methods In Enzymology (Vol. 182, Academic Press).Accordingly, this disclosure also provides the processes for obtainingthese polypeptides as well as the products obtainable and obtained bythese processes.

The polypeptides also can be obtained by chemical synthesis using acommercially available automated peptide synthesizer such as thosemanufactured by Perkin/Elmer/Applied Biosystems, Inc., Model 430A or431A, Foster City, Calif., USA. The synthesized polypeptide can beprecipitated and further purified, for example by high performanceliquid chromatography (HPLC). Accordingly, this disclosure also providesa process for chemically synthesizing the proteins disclosed herein byproviding the sequence of the protein and reagents, such as amino acidsand enzymes and linking together the amino acids in the properorientation and linear sequence.

Alternatively, the proteins and polypeptides can be obtained bywell-known recombinant methods as described, for example, in Sambrook etal. (1989) supra, using a host cell and vector systems described herein.

Also provided by this application are the polypeptides described hereinconjugated to a detectable agent for use in the diagnostic methods. Forexample, detectably labeled polypeptides can be bound to a column andused for the detection and purification of antibodies. They also areuseful as immunogens for the production of antibodies as describedbelow. The polypeptides disclosed herein are useful in an in vitro assaysystem to screen for agents or drugs, which modulate cellular processes.

It is well known to those skilled in the art that modifications can bemade to the peptides disclosed herein to provide them with alteredproperties. As used herein the term “amino acid” refers to eithernatural and/or unnatural or synthetic amino acids, including glycine andboth the D or L optical isomers, and amino acid analogs andpeptidomimetics. A peptide of three or more amino acids is commonlycalled an oligopeptide if the peptide chain is short. If the peptidechain is long, the peptide is commonly called a polypeptide or aprotein.

Peptides disclosed herein can be modified to include unnatural aminoacids. Thus, the peptides may comprise D-amino acids, a combination ofand L-amino acids, and various “designer” amino acids (e.g.,.beta.-methyl amino acids, C-alpha-methyl amino acids, andN-alpha-methyl amino acids, etc.) to convey special properties topeptides. Additionally, by assigning specific amino acids at specificcoupling steps, peptides with alpha-helices, beta. turns, beta. sheets,gamma-turns, and cyclic peptides can be generated. Generally, it isbelieved that .alpha.-helical secondary structure or random secondarystructure may be of particular use.

The polypeptides disclosed herein also can be combined with varioussolid phase carriers, such as an implant, a stent, a paste, a gel, adental implant, or a medical implant or liquid phase carriers, such asbeads, sterile or aqueous solutions, pharmaceutically acceptablecarriers, pharmaceutically acceptable polymers, liposomes, micelles,suspensions and emulsions. Examples of non-aqueous solvents includepropyl ethylene glycol, polyethylene glycol and vegetable oils. Whenused to prepare antibodies or induce an immune response in vivo, thecarriers also can include an adjuvant that is useful to non-specificallyaugment a specific immune response. A skilled artisan can easilydetermine whether an adjuvant is required and select one. However, forthe purpose of illustration only, suitable adjuvants include, but arenot limited to Freund's Complete and Incomplete, mineral salts andpolynucleotides. Other suitable adjuvants include monophosphoryl lipid A(MPL), mutant derivatives of the heat labile enterotoxin of E. coli,mutant derivatives of cholera toxin, CPG oligonucleotides, and adjuvantsderived from squalene.

This disclosure also provides a pharmaceutical composition comprising oralternatively consisting essentially of, or yet further consisting of,any of a polypeptide, analog, mutein, or fragment disclosed herein,alone or in combination with each other or other agents, such anantibiotic and an acceptable carrier or solid support. Thesecompositions are useful for various diagnostic and therapeutic methodsas described herein.

Polynucleotides

This disclosure also provides isolated or recombinant polynucleotidesencoding one or more of the above-identified isolated or recombinantpolypeptides and their respective complementary strands. Vectorscomprising the isolated or recombinant polynucleotides are furtherprovided examples of which are known in the art and briefly describedherein. In one aspect where more than one isolated or recombinantpolynucleotide is to be expressed as a single unit, the isolated orrecombinant polynucleotides can be contained within a polycistronicvector. The polynucleotides can be DNA, RNA, mRNA or interfering RNA,such as siRNA, miRNA or dsRNA.

In another aspect, this disclosure provides an interfering agent that isa polynucleotide that interferes with the binding of the DNA to apolypeptide or protein in a microbial biofilm, or a four-way junctionpolynucleotide resembling a Holliday junction, a 3 way junctionpolynucleotide resembling a replication fork, a polynucleotide that hasinherent flexibility or bent polynucleotide which can treat or inhibitDNABII polynucleotide from binding to microbial DNA as well treat,prevent or inhibit biofilm formation and associated infections anddisorders. One of skill in the art can make such polynucleotides usingthe information provided herein and knowledge of those of skill in theart. See Goodman and Kay (1999) J. Biological Chem. 274(52):37004-37011and Kamashev and Rouviere-Yaniv (2000) EMBO J. 19(23):6527-6535.

The disclosure further provides the isolated or recombinantpolynucleotide operatively linked to a promoter of RNA transcription, aswell as other regulatory sequences for replication and/or transient orstable expression of the DNA or RNA. As used herein, the term“operatively linked” means positioned in such a manner that the promoterwill direct transcription of RNA off the DNA molecule. Examples of suchpromoters are SP6, T4 and T7. In certain embodiments, cell-specificpromoters are used for cell-specific expression of the insertedpolynucleotide. Vectors which contain a promoter or a promoter/enhancer,with termination codons and selectable marker sequences, as well as acloning site into which an inserted piece of DNA can be operativelylinked to that promoter are known in the art and commercially available.For general methodology and cloning strategies, see Gene ExpressionTechnology (Goeddel ed., Academic Press, Inc. (1991)) and referencescited therein and Vectors: Essential Data Series (Gacesa and Ramji,eds., John Wiley & Sons, N.Y. (1994)) which contains maps, functionalproperties, commercial suppliers and a reference to GenEMBL accessionnumbers for various suitable vectors.

In one embodiment, polynucleotides derived from the polynucleotidesdisclosed herein encode polypeptides or proteins having diagnostic andtherapeutic utilities as described herein as well as probes to identifytranscripts of the protein that may or may not be present. These nucleicacid fragments can by prepared, for example, by restriction enzymedigestion of larger polynucleotides and then labeled with a detectablemarker. Alternatively, random fragments can be generated using nicktranslation of the molecule. For methodology for the preparation andlabeling of such fragments, see, Sambrook et al. (1989) supra.

Expression vectors containing these nucleic acids are useful to obtainhost vector systems to produce proteins and polypeptides. It is impliedthat these expression vectors must be replicable in the host organismseither as episomes or as an integral part of the chromosomal DNA.Non-limiting examples of suitable expression vectors include plasmids,yeast vectors, viral vectors and liposomes. Adenoviral vectors areparticularly useful for introducing genes into tissues in vivo becauseof their high levels of expression and efficient transformation of cellsboth in vitro and in vivo. When a nucleic acid is inserted into asuitable host cell, e.g., a prokaryotic or a eukaryotic cell and thehost cell replicates, the protein can be recombinantly produced.Suitable host cells will depend on the vector and can include mammaliancells, animal cells, human cells, simian cells, insect cells, yeastcells, and bacterial cells constructed using known methods. See,Sambrook et al. (1989) supra. In addition to the use of viral vector forinsertion of exogenous nucleic acid into cells, the nucleic acid can beinserted into the host cell by methods known in the art such astransformation for bacterial cells; transfection using calcium phosphateprecipitation for mammalian cells; or DEAE-dextran; electroporation; ormicroinjection. See, Sambrook et al. (1989) supra, for methodology.Thus, this disclosure also provides a host cell, e.g., a mammalian cell,an animal cell (rat or mouse), a human cell, or a prokaryotic cell suchas a bacterial cell, containing a polynucleotide encoding a protein orpolypeptide or antibody.

A polynucleotide can comprise modified nucleotides, such as methylatednucleotides and nucleotide analogs. If present, modifications to thenucleotide structure can be imparted before or after assembly of thepolynucleotide. The sequence of nucleotides can be interrupted bynon-nucleotide components. A polynucleotide can be further modifiedafter polymerization, such as by conjugation with a labeling component.The term also refers to both double- and single-stranded molecules.Unless otherwise specified or required, any embodiment disclosed hereinthat is a polynucleotide encompasses both the double-stranded form andeach of two complementary single-stranded forms known or predicted tomake up the double-stranded form.

When the vectors are used for gene therapy in vivo or ex vivo, apharmaceutically acceptable vector, such as a replication-incompetentretroviral or adenoviral vector, are exemplary (but non-limiting) andmay be of particular use. Pharmaceutically acceptable vectors containingthe nucleic acids disclosed herein can be further modified for transientor stable expression of the inserted polynucleotide. As used herein, theterm “pharmaceutically acceptable vector” includes, but is not limitedto, a vector or delivery vehicle having the ability to selectivelytarget and introduce the nucleic acid into dividing cells. An example ofsuch a vector is a “replication-incompetent” vector defined by itsinability to produce viral proteins, precluding spread of the vector inthe infected host cell. An example of a replication-incompetentretroviral vector is LNL6 (Miller et al. (1989) BioTechniques7:980-990). The methodology of using replication-incompetentretroviruses for retroviral-mediated gene transfer of gene markers hasbeen established. (Bordignon (1989) PNAS USA 86:8912-8952; Culver (1991)PNAS USA 88:3155; and Rill (1991) Blood 79(10):2694-2700).

This disclosure also provides genetically modified cells that containand/or express the polynucleotides disclosed herein. The geneticallymodified cells can be produced by insertion of upstream regulatorysequences such as promoters or gene activators (see, U.S. Pat. No.5,733,761).

The polynucleotides can be conjugated to a detectable marker, e.g., anenzymatic label or a radioisotope for detection of nucleic acid and/orexpression of the gene in a cell. A wide variety of appropriatedetectable markers are known in the art, including fluorescent,radioactive, enzymatic or other ligands, such as avidin/biotin, whichare capable of giving a detectable signal. In one aspect, one willlikely desire to employ a fluorescent label or an enzyme tag, such asurease, alkaline phosphatase or peroxidase, instead of radioactive orother environmentally undesirable reagents. In the case of enzyme tags,calorimetric indicator substrates can be employed to provide a meansvisible to the human eye or spectrophotometrically, to identify specifichybridization with complementary nucleic acid-containing samples. Thus,this disclosure further provides a method for detecting asingle-stranded polynucleotide or its complement, by contacting targetsingle-stranded polynucleotide with a labeled, single-strandedpolynucleotide (a probe) which is a portion of the polynucleotidedisclosed herein under conditions permitting hybridization (optionallymoderately stringent hybridization conditions) of complementarysingle-stranded polynucleotides, or optionally, under highly stringenthybridization conditions. Hybridized polynucleotide pairs are separatedfrom un-hybridized, single-stranded polynucleotides. The hybridizedpolynucleotide pairs are detected using methods known to those of skillin the art and set forth, for example, in Sambrook et al. (1989) supra.

The polynucleotide embodied in this disclosure can be obtained usingchemical synthesis, recombinant cloning methods, PCR, or any combinationthereof. Methods of chemical polynucleotide synthesis are known in theart and need not be described in detail herein. One of skill in the artcan use the sequence data provided herein to obtain a desiredpolynucleotide by employing a DNA synthesizer or ordering from acommercial service.

The polynucleotides disclosed herein can be isolated or replicated usingPCR. The PCR technology is the subject matter of U.S. Pat. Nos.4,683,195; 4,800,159; 4,754,065; and 4,683,202 and described in PCR: ThePolymerase Chain Reaction (Mullis et al. eds., Birkhauser Press, Boston(199.4)) or MacPherson et al. (1991) and (1995) supra, and referencescited therein. Alternatively, one of skill in the art can use thesequences provided herein and a commercial DNA synthesizer to replicatethe DNA. Accordingly, this disclosure also provides a process forobtaining the polynucleotides disclosed herein by providing the linearsequence of the polynucleotide, nucleotides, appropriate primermolecules, chemicals such as enzymes and instructions for theirreplication and chemically replicating or linking the nucleotides in theproper orientation to obtain the polynucleotides. In a separateembodiment, these polynucleotides are further isolated. Still further,one of skill in the art can insert the poly-nucleotide into a suitablereplication vector and insert the vector into a suitable host cell(prokaryotic or eukaryotic) for replication and amplification. The DNAso amplified can be isolated from the cell by methods known to those ofskill in the art. A process for obtaining polynucleotides by this methodis further provided herein as well as the polynucleotides so obtained.

RNA can be obtained by first inserting a DNA polynucleotide into asuitable host cell. The DNA can be delivered by any appropriate method,e.g., by the use of an appropriate gene delivery vehicle (e.g.,liposome, plasmid or vector) or by electroporation. When the cellreplicates and the DNA is transcribed into RNA; the RNA can then beisolated using methods known to those of skill in the art, for example,as set forth in Sambrook et al. (1989) supra. For instance, mRNA can beisolated using various lytic enzymes or chemical solutions according tothe procedures set forth in Sambrook et al. (1989) supra, or extractedby nucleic-acid-binding resins following the accompanying instructionsprovided by manufactures.

Polynucleotides exhibiting sequence complementarity or homology to apolynucleotide disclosed herein are useful as hybridization probes or asan equivalent of the specific polynucleotides identified herein. Sincethe full coding sequence of the transcript is known, any portion of thissequence or homologous sequences can be used in the methods disclosedherein.

It is known in the art that a “perfectly matched” probe is not neededfor a specific hybridization. Minor changes in probe sequence achievedby substitution, deletion or insertion of a small number of bases do notaffect the hybridization specificity. In general, as much as 20%base-pair mismatch (when optimally aligned) can be tolerated. In someembodiments, a probe useful for detecting the aforementioned mRNA is atleast about 80% identical to the homologous region. In some embodiments,the probe is 85% identical to the corresponding gene sequence afteralignment of the homologous region; in some embodiments, it exhibits 90%identity.

These probes can be used in radioassays (e.g., Southern and Northernblot analysis) to detect, prognose, diagnose or monitor various cells ortissues containing these cells. The probes also can be attached to asolid support or an array such as a chip for use in high throughputscreening assays for the detection of expression of the genecorresponding a polynucleotide disclosed herein. Accordingly, thisdisclosure also provides a probe comprising or corresponding to apolynucleotide disclosed herein, or its equivalent, or its complement,or a fragment thereof, attached to a solid support for use in highthroughput screens.

The total size of fragment, as well as the size of the complementarystretches, will depend on the intended use or application of theparticular nucleic acid segment. Smaller fragments will generally finduse in hybridization embodiments, wherein the length of thecomplementary region may be varied, such as between at least 5 to 10 toabout 100 nucleotides, or even full length according to thecomplementary sequences one wishes to detect.

Nucleotide probes having complementary sequences over stretches greaterthan 5 to 10 nucleotides in length are generally well suited, so as toincrease stability and selectivity of the hybrid, and thereby improvingthe specificity of particular hybrid molecules obtained. In certainembodiments, one can design polynucleotides having gene-complementarystretches of 10 or more or more than 50 nucleotides in length, or evenlonger where desired. Such fragments may be readily prepared by, forexample, directly synthesizing the fragment by chemical means, byapplication of nucleic acid reproduction technology, such as the PCRtechnology with two priming oligonucleotides as described in U.S. Pat.No. 4,603,102 or by introducing selected sequences into recombinantvectors for recombinant production. In one aspect, a probe is about50-75 or more alternatively, 50-100, nucleotides in length.

The polynucleotides of the present disclosure can serve as primers forthe detection of genes or gene transcripts that are expressed in cellsdescribed herein. In this context, amplification means any methodemploying a primer-dependent polymerase capable of replicating a targetsequence with reasonable fidelity. Amplification may be carried out bynatural or recombinant DNA-polymerases such as T7 DNA polymerase, Klenowfragment of E. coli DNA polymerase, and reverse transcriptase. Forillustration purposes only, a primer is the same length as thatidentified for probes.

One method to amplify polynucleotides is PCR and kits for PCRamplification are commercially available. After amplification, theresulting DNA fragments can be detected by any appropriate method knownin the art, e.g., by agarose gel electrophoresis followed byvisualization with ethidium bromide staining and ultravioletillumination.

Methods for administering an effective amount of a gene delivery vectoror vehicle to a cell have been developed and are known to those skilledin the art and described herein. Methods for detecting gene expressionin a cell are known in the art and include techniques such as inhybridization to DNA microarrays, in situ hybridization, PCR, RNaseprotection assays and Northern blot analysis. Such methods are useful todetect and quantify expression of the gene in a cell. Alternativelyexpression of the encoded polypeptide can be detected by variousmethods. In particular it is useful to prepare polyclonal or monoclonalantibodies that are specifically reactive with the target polypeptide.Such antibodies are useful for visualizing cells that express thepolypeptide using techniques such as immunohistology, ELISA, and Westernblotting. These techniques can be used to determine expression level ofthe expressed polynucleotide.

Antibodies and Derivatives Thereof

This disclosure also provides an antibody that binds and/or specificallyrecognizes and binds an isolated polypeptide for use in the methodsdisclosed herein. The antibody can be any of the various antibodiesdescribed herein, non-limiting, examples of such include a polyclonalantibody, a monoclonal antibody, a chimeric antibody, a human antibody,a veneered antibody, a diabody, a humanized antibody, an antibodyderivative, a recombinant humanized antibody, or a derivative orfragment of each thereof. In one aspect, the fragment comprises, oralternatively consists essentially of, or yet further consists of theCDR of the antibody. In one aspect, the antibody is detectably labeledor further comprises a detectable label conjugated to it. Also providedis a hybridoma cell line that produces a monoclonal antibody disclosedherein. Compositions comprising or alternatively consisting essentiallyof or yet further, consisting of one or more of the above embodimentsare further provided herein. Further provided are polynucleotides thatencode the amino acid sequence of the antibodies and fragments as wellas methods to produce recombinantly or chemically synthesize theantibody polypeptides and fragments thereof. The antibody polypeptidescan be produced in a eukaryotic or prokaryotic cell, or by other methodsknown in the art and described herein.

Examples of CDR sequences include without limitation comprise, consistessentially of, or yet further consist of, the following: the heavychain variable region of the antibody or a fragment thereof comprises,or alternatively consists essentially of, or yet further consists of,the polypeptide encoded by the below polynucleotide sequence:

SEQ ID NO. (358): Non-limiting exemplary heavy chain variable regionnucleotide sequence, IhfA5 fragment

GAGGTGCAGCTGCAGGAGTCTGGACCTGGCCTGGTGACGCCCTCACAGAGCCTGTCCATGACTTGCACTGTCTCTGGGTTTTCATTAACCAGCTATAGTGTACACTGGGTTCGCCAGCCTCCAGGAAAGAGTCTGGAGTGGCTGGGAGTAATATGGGCTGGTGGAAGCACAAATTATAATTCGGCTCTCATGTCCAGACTGAGCATCAGCAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGGACAGTCTGCAAACTGATGACACAGCCATATACTACTGTGCCAGAGAGGACTCCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

In some embodiments, the heavy chain variable region of the antibody ora fragment thereof comprises, or alternatively consists essentially of,or yet further consists of, the amino acid sequence:

SEQ ID NO. (359): Non-limiting exemplary heavy chain variable regionamino acid sequence, IhfA5 fragment

EVQLQESGPGLVTPSQSLSMTCTVSGFSLTSYSVHWVRQPPGKSLEWLGVIWAGGSTNYNSALMSRLSISKDNSKSQVFLKMDSLQTDDTAIYYCARE DSWGQGTSVTVSS

In some embodiments, the light chain variable region of the antibody ora fragment thereof comprises, or alternatively consists essentially of,or yet further consists of, the polypeptide encoded by the belowpolynucleotide sequence:

SEQ ID NO. (360): Non-limiting exemplary light chain variable regionnucleotide sequence, IhfA5 fragment

GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCCACGTTCGGAGGGGGGACCAAGTTGGAAATAAAA

In some embodiments, the light chain variable region of the antibody ora fragment thereof comprises, or alternatively consists essentially of,or yet further consists of, the amino acid sequence:

SEQ ID NO. (361): Non-limiting exemplary light chain variable regionamino acid sequence, IhfA5 fragment

DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPTFG GGTKLEIK

In some embodiments the antibodies disclosed herein comprise, oralternatively consists essentially of, or yet further consists of, thepolypeptide encoded by the polynucleotide sequence comprising one ormore of the CDR sequences listed herein below:

SEQ ID NO. (362) Partial non-limiting exemplary CDRH1 sequence, IhfA5fragment FSLTSYS

SEQ ID NO. (363) Partial non-limiting exemplary CDRH1 sequence, IhfA5fragment FSLTSYSV

SEQ ID NO. (364): Partial non-limiting exemplary CDRH1 sequence, IhfA5fragment

FSLTSYSVH

SEQ ID NO. (365): Partial non-limiting exemplary CDRH1 sequence, IhfA5fragment

GFSLTSYS

SEQ ID NO. (366): Partial non-limiting exemplary CDRH2 sequence, IhfA5fragment

IWAGGST

SEQ ID NO. (367): Partial non-limiting exemplary CDRH2 sequence, IhfA5fragment

VIWAGGST

SEQ ID NO. (368): Partial non-limiting exemplary CDRH2 sequence, IhfA5fragment

GVIWAGGST

SEQ ID NO. (369): Partial non-limiting exemplary CDRH2 sequence, IhfA5fragment

LGVIWAGGST

SEQ ID NO. (370): Partial non-limiting exemplary CDRH2 sequence, IhfA5fragment

WLGVIWAGGST

SEQ ID NO. (371): Partial non-limiting exemplary CDRH2 sequence, IhfA5fragment

IWAGGSTN

SEQ ID NO. (372): Partial non-limiting exemplary CDRH2 sequence, IhfA5fragment

VIWAGGSTN

SEQ ID NO. (373): Partial non-limiting exemplary CDRH2 sequence, IhfA5fragment

GVIWAGGSTN

SEQ ID NO. (374): Partial non-limiting exemplary CDRH2 sequence, IhfA5fragment

LGVIWAGGSTN

SEQ ID NO. (375): Partial non-limiting exemplary CDRH2 sequence, IhfA5fragment

WLGVIWAGGSTN

SEQ ID NO. (376): Partial non-limiting exemplary CDRH2 sequence, IhfA5fragment

IWAGGSTNY

SEQ ID NO. (377): Partial non-limiting exemplary CDRH2 sequence, IhfA5fragment

VIWAGGSTNY

SEQ ID NO. (378): Partial non-limiting exemplary CDRH2 sequence, IhfA5fragment

GVIWAGGSTNY

SEQ ID NO. (379): Partial non-limiting exemplary CDRH2 sequence, IhfA5fragment

LGVIWAGGSTNY

SEQ ID NO. (380): Partial non-limiting exemplary CDRH2 sequence, IhfA5fragment

WLGVIWAGGSTNY

SEQ ID NO. (381): Partial non-limiting exemplary CDRH3 sequence, IhfA5fragment REDS

SEQ ID NO. (382): Partial non-limiting exemplary CDRH3 sequence, IhfA5fragment AREDS

SEQ ID NO. (383): Partial non-limiting exemplary CDRL1 sequence, IhfA5fragment QNVGTN

SEQ ID NO. (384): Partial non-limiting exemplary CDRL1 sequence, IhfA5fragment QNVGTNV

SEQ ID NO. (385): Partial non-limiting exemplary CDRL1 sequence, IhfA5fragment QNVGTNVA

SEQ ID NO. (386): Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment SAS

SEQ ID NO. (387): Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment YSAS

SEQ ID NO. (388): Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment IYSAS

SEQ ID NO. (389) Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment LIYSAS

SEQ ID NO. (390): Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment ALIYSAS

SEQ ID NO. (391) Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment SASY

SEQ ID NO. (392): Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment YSASY

SEQ ID NO. (393): Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment IYSASY

SEQ ID NO. (394): Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment LIYSASY

SEQ ID NO. (395): Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment ALIYSASY

SEQ ID NO (396): Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment SASYR

SEQ ID NO. (397): Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment YSASYR

SEQ ID NO. (398) Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment IYSASYR

SEQ ID NO. (399): Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment LIYSASYR

SEQ ID NO. (400): Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment ALIYSASYR

SEQ ID NO. (401): Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment SASYRY

SEQ ID NO. (402): Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment YSASYRY

SEQ ID NO. (403): Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment IYSASYRY

SEQ ID NO. (404): Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment LIYSASYRY

SEQ ID NO. (405): Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment ALIYSASYRY

SEQ ID NO. (406): Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment SASYRYS

SEQ ID NO. (407): Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment YSASYRYS

SEQ ID NO. (408): Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment IYSASYRYS

SEQ ID NO. (409): Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment LIYSASYRYS

SEQ ID NO. (410): Partial non-limiting exemplary CDRL2 sequence, IhfA5fragment ALIYSASYRYS

SEQ ID NO. (411): Partial non-limiting exemplary CDRL3 sequence, IhfA5fragment QQYNSYP

SEQ ID NO. (412): Partial non-limiting exemplary CDRL3 sequence, IhfA5fragment QQYNSYPT

Antibodies can be generated using conventional techniques known in theart and are well-described in the literature. Several methodologiesexist for production of polyclonal antibodies. For example, polyclonalantibodies are typically produced by immunization of a suitable mammalsuch as, but not limited to, chickens, goats, guinea pigs, hamsters,horses, mice, rats, and rabbits. An antigen is injected into the mammal,induces the B-lymphocytes to produce immunoglobulins specific for theantigen. Immunoglobulins may be purified from the mammal's serum.Antibodies specific to DNABII polypeptides, e.g., IHFα and IHFβ can begenerated by injection of polypeptides corresponding to differentepitopes of IHFα and IHFβ. For example, antibodies can be generatedusing the 20 amino acids of each subunit such as TFRPGQKLKSRVENASPKDE(SEQ ID NO:34) for IHFα and KYVPHFKPGKELRDRANIYG (SEQ ID NO:35) for IHFβor A1 to A6 or B1 to B6 (See FIG. 18 ). Antibodies specific to the “tip”or “tail” portion of the DNABII protein or polypeptide can be generated.Additional non-limiting examples include without limitation apolypeptide fragment that comprises one or more of MATITKLDIIEYLSDKYHLS(also referred to herein as hIFA1; (SEQ ID NO. 348));KYHLSKQDTKNVVENFLEEI (also referred to herein as hIFA2; (SEQ ID NO.349)); FLEEIRLSLESGQDVKLSGF (also referred to herein as hIFA3; (SEQ IDNO. 350)); KLSGFGNFELRDKSSRPGRN (also referred to herein as hIFA4; (SEQID NO. 351)); RPGRNPKTGDVVPVSARRVV (also referred to herein as hIFA5;(SEQ ID NO. 352)); ARRVVTFKPGQKLRARVEKTK (also referred to herein ashIFA6; (SEQ ID NO. 353)); or an equivalent thereof. Other exemplaryantibodies include monoclonal antibodies that specifically recognize andbind Haemophilus influenzae IhfA fragment A5 (SEQ ID NO. 352)). Thehybridoma cell lines that produce monoclonal antibodies thatspecifically recognize and bind Haemophilus influenzae IhfA fragment A5(SEQ ID NO. 352)) and IhfB4 (RGFGSFSLHHRQPRLGRNPK (also referred to B4(SEQ ID NO. 345)); were deposited with American Type Culture Collection(ATCC) under Accession Numbers (IhfA5 (Accession No. PTA-122334)) and(IhfB4 (Accession No. PTA-122336)), pursuant to the provisions of theBudapest Treaty on Jul. 30, 2015; the respective hybridoma cell linesdesignated: IhfA5-NTHI 14 GB.F5.G6; and IhfB4-NTHI 4EII.E5.G2. Furthernon-limiting exemplary antibodies include those that specificallyrecognize and bind Haemophilus influenzae IhfA fragment A3 (SEQ ID NO.350), IhfB fragment B2 (SEQ ID NO. 343) produced by hybridoma cell linesIhfA3 NTHI 9B10.F2.H3 and IhfB2 NTHI 7A4.E4.G4, respectively.

Further, non-limiting exemplary antibodies that fall within the scope ofthis disclosure include but are not limited to those disclosed in nowabandoned U.S. Ser. No. 14/497,147, filed Sep. 25, 2014; U.S. Ser. No.14/668,767, filed Mar. 25, 2015; and U.S. Ser. No. 14/789,842, filedJul. 1, 2015, and provided under the following designations: TRL295,TRL299, TRL1012, TRL1068, TRL1070, TRL1087, TRL1215, TRL1216, TRL1218,TRL1230, TRL1232, TRL1242, TRL1245, TRL1330, TRL1335, TRL1337, TRL1338,TRL1341, TRL1347, and TRL1361, the heavy chain and light chain sequencesof which are provided herein below.

TRL295 HC, SEQ ID NO: 413QVQLVESGGGLVQPGGSLRLSCAASGFPFSSYAMSWVRQAPGKGLEWVSAISGNGADSYYADSVKGRFTTSRDKSKNTVYLQMNRLRAEDTAVYYCAKDMRRYHYDSSGLHFWG QGTLVTVSSTRL295 LC, SEQ ID NO: 414DIELTQAPSVSVYPGQTARITCSGDALPKQYAYWYQQKPGQAPVVVIYKDSERPSGISERFSGSSSGTTVTLTISGVQAGDEADYYCQSVDTSVSYYVVFGGGTKLTVL TRL299 HC,SEQ ID NO: 415QVQLVQSGGGLVQPGGSLRLSCAASGFTFSHYNMNWVRQAPGKGPEWVSYISSGSDIIYYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARALDRDGFDIWGQGTMVTVSS TRL299 LC,SEQ ID NO: 416DIVLTQSPSSLSASVGDKVTITCRASQSISTFLNWYQHKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPTFGQGTKVEIK TRL1012 HC, SEQ ID NO: 417QVQLVESGGGLVQPGGSLRLSCAASGFPFSSYAMSWVRQAPGKGLEWVSAISGNGADSYYADSVKGRFTTSRDKSKNTVYLQMNRLRAEDTAVYYCAKDMRRYHYDSSGLHFWG QGTLVTVSSTRL1012 LC, SEQ ID NO: 418DIMLTQPPSVSAAPGQKVTISCSGSSSNIGTNYVSWFQQVPGTAPKFLIYDNYKRPSETPDRFSGSKSGTSATLDITGLQTGDEANYYCATWDSSLSAWVFGGGTKVTVL TRL1068 HC,SEQ ID NO: 419QVQLVESGPGLVKPSETLSLTCRVSGDSNRPSYWSWIRQAPGKAMEWIGYVYDSGVTIYNPSLKGRVTISLDTSKTRFSLKLTSVIAADTAVYYCARERFDRTSYKSWWGQGTQVTVSS TRL1068 LC,SEQ ID NO: 420DIVLTQAPGTLSLSPGDRATLSCRASQRLGGTSLAWYQHRSGQAPRLILYGTSNRATDTPDRFSGSGSGTDFVLTISSLEPEDFAVYYCQQYGSPPYTFGQGTTLDIK TRL1070 HC,SEQ ID NO: 421QVQLVQSGGTLVQPGGSLRLSCAASGFTFSYYSMSWVRQAPGKGLEWVANIKHDGTERNYVDSVKGRFTISRDNSEKSLYLQMNSLRAEDTAVYYCAKYYYGAGTNYPLKYWGQG TRVTVSSTRL1070 LC, SEQ ID NO: 422DILMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPLTFGGGTKVEIKR TRL1087 HC,SEQ ID NO: 423QVQLLESGPGLVRPSDTLSLTCTFSADLSTNAYWTWIRQPPGKGLEWIGYMSHSGGRDYNPSFNRRVTISVDTSKNQVFLRLTSVTSADTAVYFCVREVGSYYDYWGQGILVTVSS TRL1087 LC,SEQ ID NO: 424DIEMTQSPSSLSASVGDRITITCRASQGISTWLAWYQQKPGKAPKSLIFSTSSLHSGVPSKFSGSGSGTDFTLTITNLQPEDFATYYCQQKWETPYSFGQGTKLDMIR TRL1215 HC,SEQ ID NO: 425 QVQLVESGTEVKNPGASVKVSCTASGYKFDEYGVSWVRQSPGQGLEWMGWISVYNGKTNYSQNFQGRLTLTTETSTDTAYMELTSLRPDDTAVYYCATDKNWFDPWGPGTLVTVSS TRL1215 LC,SEQ ID NO: 426DIVMTQSPSASGSPGQSITISCTGTNTDYNYVSWYQHHPGKAPKVIIYDVKKRPSGVPSRFSGSRSGNTATLTVSGLQTEDEADYYCVSYADNNHYVFGSGTKVTVL TRL1216 HC,SEQ ID NO: 427 QVQLVESGGGVVQPGGSLRVSCAASAFSFRDYGIHWVRQAPGKGLQWVAVISHDGGKKFYADSVRGRFTISRDNSENTLYLQMNSLRSDDTAVYYCARLVASCSGSTCTTQPAAFD IWGPGTLVTVSSTRL1216 LC, SEQ ID NO: 428DIMLTQPPSVSVSPGQTARITCSGDALPKKYTYWYQQKSGQAPVLLIYEDRKRPSEIPERFSAFTSWTTATLTITGAQVRDEADYYCYSTDISGDIGVFGGGTKLTVL TRL1218 HC,SEQ ID NO: 429 QVQLLESGADMVQPGRSLRLSCAASGFNFRTYAMHWVRQAPGKGLEWVAVMSHDGYTKYYSDSVRGQFTISRDNSKNTLYLQMNNLRPDDTAIYYCARGLTGLSVGFDYWGQGT LVTVSSTRL1218 LC, SEQ ID NO: 430DIVLTQSASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVTTRPSGVSDRFSGSKSGNTASLTISGLQAEDEADYYCSSYSSGSTPALFGGGTQLTVL TRL1230 HC,SEQ ID NO: 431QVQLVQSGGGLVKPGGSLRLSCGASGFNLSSYSMNWVRQAPGKGLEWVSSISSRSSYIYYADSVQGRFTISRDNAKNSLYLQMNSLRAEDTAIYYCARVSPSTYYYYGMDVWGQGT TVTVSSTRL1230 LC, SEQ ID NO: 432DIVLTQPSSVSVSPGQTARITCSGDELPKQYAYWYQQKPGQAPVLVIYKDNERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYCQSADSSGTYVVFGGGTKLTVL TRL1232 HC,SEQ ID NO: 433 QVQLVESGAEVKKPGALVKVSCKASGYTFSGYYMHWVRQAPGQGLEWMGWINPKSGGTKYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYFCARGGPSNLERFLERLQPRYSYDDKYAMDVWGQGTTVTVSS TRL1232 LC, SEQ ID NO: 434DIVMTQSPGTLSLSPGARATLSCRASQSVSSIYLAWYQQKPGQAPRLLIFGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPYTFGQGTKLEIKR TRL1242 HC,SEQ ID NO: 435QVQLVQSGTEVKKPGESLKISCEGSRYNFARYWIGWVRQMPGKGLDWMGIIYPGDSDTRYSPSFQGQVSISADKSISTAYLQWNSLKASDTAMYYCARLGSELGVVSDYYFDSWGQ GTLVTVSSTRL1242 LC, SEQ ID NO: 436DIVLTQSPDSLAVSLGERATINCKSSQSVLDRSNNKNCVAWYQQKPGQPPKLLIYRAATRESGVPDRFSGSGSGTDFSLTISSLQAEDVAVYFCQQYYSIPNTFGQGTKLEIKR TRL1245 HC,SEQ ID NO: 437QVQLVESGGGLVKAGGSLRLSCVASGFTFSDYYMSWIRQAPGKGLEWISFISSSGDTIFYADSVKGRFTVSRDSAKNSLYLQMNSLKVEDTAVYYCARKGVSDEELLRFWGQGTLVTVSS TRL1245 LC,SEQ ID NO: 438DIVLTQDPSVSVSPGQTARITCSGDALPKKYAYWYQQKSGQAPVLVIYEDTKRPSGIPERFSGSSSGTVATLTISGAQVEDEADYYCYSTDSSGNQRVFGGGTKLTVL TRL1330 HC,SEQ ID NO: 439 QVQLVESGTEVKNPGASVKVSCTASGYKFDEYGVSWVRQSPGQGLEWMGWISVYNGKTNYSQNFQGRLTLTTETSTDTAYMELTSLRPDDTAVYYCATDKNWFDPWGPGTLVTVSS TRL1330 LC,SEQ ID NO: 440DIVLTQSPSASGSPGQSITISCTGTNTDYNYVSWYQHHPGKAPKVIIYDVKKRPSGVPSRFSGSRSGNTATLTVSGLQTEDEADYYCVSYADNNHYVFGSGTKVTVL TRL1335 HC,SEQ ID NO: 441QVQLVESGAEVKKPGESLKISCKGSGYNFTSYWIGWVRQMPGKGLEWMGVIYPDDSDTRYSPSFKGQVTISADKSISTAFLQWSSLKASDTAVYHCARPPDSWGQGTLVTVSS TRL1335 LC,SEQ ID NO: 442DIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGLAPRLLIVGASNRATGIPARFSGSGSGTEFTLTISSLQSEDFAFYYCQQYNNWPFTFGPGTKVDVKR TRL1337 HC,SEQ ID NO: 443QVQLLESGPGLVKPSETPSLTCTVSGGSIRSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDMSKNQFSLKLSSVTAADTAMYYCARVYGGSGSYDFDYWGQGTLVTVSS TRL1337 LC,SEQ ID NO: 444DIVLTQSPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQQLPGKAPKLMIYEVTKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCSSFAGSNNHVVFGGGTKLTVL TRL1338 HC,SEQ ID NO: 445QVQLTLRESGPTLVKPTQTLTLTCTFSGFSLSTNGVGVGWIRQPPGKALEWLAIIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTLTNMDPVDTGTYYCAHILGASNYWTGYLRYYFD YWGQGTLVTVSTTRL1338 LC, SEQ ID NO: 446DIEMTQSPSVSVSPGQTARITCSGEPLAKQYAYWYQQKSGQAPVVVIYKDTERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYHCESGDSSGTYPVFGGGTKLTVL TRL1341 HC,SEQ ID NO: 447QVQLQESGGGLVQPGGSLKLSCAASGFIFSGSTMHWVRQASGKGLEWVGRIRSKTNNYATAYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCISLPGGYSSGQGTLVTVSS TRL1341 LC,SEQ ID NO: 448DIMLTQPPSVSVSPGQTARITCSGDALPKKYTYWYQQKSGQAPVLVIYEDSKRPSEIPERFSAFTSWTTATLTITGAQVGDEADYYCYSTDITGDIGVFGGGTKLTVL TRL1347 HC,SEQ ID NO: 449QVQLVQSGGGLVQPGGSLKVSCVGSGFTFSASTIHWVRQASGKGLEWVGRIRSKANNYATVSAASLKGRFTISRDDSKNTAYLQVNSLKIEDTAIYYCTRPTACGDRVCWHGAWGQ GTQVTVSPTRL1347 LC, SEQ ID NO: 450DIVLTQSPSASGTPGQRVTISCSGSRSNLGNNNVNWYQQLPGTAPKLLIFDNNERPSGVPGRFSGSKSGTSASLAISGLRSEDEADYYCASWDDSLNGWVFGGGTKVTVL TRL1361 HC,SEQ ID NO: 451 QVQLVESGGGLAQPGGSLRLSCAASGFIFNTYAMGWVRQAPGKGLEWVSTVSAPGAGTYYTDSVKGRFIISRDNSKNILYLQMNRLRVEDTAVYYCARDQGGPAVAGARIFDYWG QGALVTVSSTRL1361 LC, SEQ ID NO: 452DIVLTQSPLSLSVTPGQPASISCKSSQSLLRSDGKTYLCWYLQKPGQPPQLLIYEVSNRVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSIQLRTFGQGTKVEIKR

Further non-limiting exemplary antibodies contemplated are antibodieshaving the CDR regions of the above-noted TRL295, TRL299, TRL1012,TRL1068, TRL1070, TRL1087, TRL1215, TRL1216, TRL1218, TRL1230, TRL1232,TRL1242, TRL1245, TRL1330, TRL1335, TRL1337, TRL1338, TRL1341, TRL1347,and TRL1361. Methods of identifying CDR regions are known in the art.For example, CDR predictions may be made based on the heavy and/or lightchain sequences—e.g. based on the Kabat, Clothia, AbM, or contactdefinitions of CDR specificity; details of these CDR prediction methodsare known in the art (see, e.g., bioinforg.uk/abs/#cdrid), utilizing theCDR prediction algorithms provided by the Ofran Lab (Paratome availableat ofranservices.biu.acil/site/services/paratome/index.html) and GreenMountain Antibodies' CDR prediction program, and/or other commerciallyavailable resources.

The disclosed antibodies are known to bind to DNABII proteins. Forexample. TRL295 is reported to bind to residues 61-80 of Haemophilusinfluenzae IHF—also known as IhfA5. TRL1068 and TRL1337 are reported toexhibit high affinity binding for the amino acid fragmentsAARKGRNPQTGKEID (SEQ ID NO: 453) and KGRNPQTGKEIDIPA (SEQ ID NO: 454) ofStaphylococcus aureus HU (SEQ ID NO: 59). TRL1330 is reported to exhibithigh affinity binding for the amino acid fragment KGRNPQTGKEIDI (SEQ IDNO: 455) of Staphylococcus aureus HU (SEQ ID NO: 59). TRL1338 isreported to exhibit high affinity binding for the amino acid fragmentVPAFKAGKALKDAVK (SEQ ID NO: 456) of Staphylococcus aureus HU (SEQ ID NO:59). TRL1361 is reported to exhibit high affinity binding for the aminoacid fragments SLAKGEKVQLIGFGN (SEQ ID NO: 457) and KGEKVQLIGFGNFEV (SEQID NO: 458) of Staphylococcus aureus HU (SEQ ID NO: 59).

Variations of this methodology include modification of adjuvants, routesand site of administration, injection volumes per site and the number ofsites per animal for optimal production and humane treatment of theanimal. For example, adjuvants typically are used to improve or enhancean immune response to antigens. Most adjuvants provide for an injectionsite antigen depot, which allows for a stow release of antigen intodraining lymph nodes. Other adjuvants include surfactants which promoteconcentration of protein antigen molecules over a large surface area andimmunostimulatory molecules. Non-limiting examples of adjuvants forpolyclonal antibody generation include Freund's adjuvants, Ribi adjuvantsystem, and Titermax. Polyclonal antibodies can be generated usingmethods known in the art some of which are described in U.S. Pat. Nos.7,279,559; 7,119,179; 7,060,800; 6,709,659; 6,656,746; 6,322,788;5,686,073; and 5,670,153.

Monoclonal antibodies can be generated using conventional hybridomatechniques known in the art and well-described in the literature. Forexample, a hybridoma is produced by fusing a suitable immortal cell line(e.g., a myeloma cell line such as, but not limited to, Sp2/0,Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, P3X63Ag8,653, Sp2 SA3, Sp2 MAI,Sp2 SS1, Sp2 SA5, U397, MIA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI,K-562, COS, RAH, NIH 313, HL-60, MLA 144, NAMAIWA, NEURO 2A, CHO,PerC.6, YB2/O) or the like, or heteromyelomas, fusion products thereof,or any cell or fusion cell derived there from, or any other suitablecell line as known in the art (see, those at the following webaddresses, e.g., atcc.org, lifetech.com, last accessed on Nov. 26,2007), with antibody producing cells, such as, but not limited to,isolated or cloned spleen, peripheral blood, lymph, tonsil, or otherimmune or B cell containing cells, or any other cells expressing heavyor light chain constant or variable or framework or CDR sequences,either as endogenous or heterologous nucleic acid, as recombinant orendogenous, viral, bacterial, algal, prokaryotic, amphibian, insect,reptilian, fish, mammalian, rodent, equine, ovine, goat, sheep, primate,eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA,chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triplestranded, hybridized, and the like or any combination thereof. Antibodyproducing cells can also be obtained from the peripheral blood or, inparticular embodiments, the spleen or lymph nodes, of humans or othersuitable animals that have been immunized with the antigen of interestand then screened for the activity of interest. Any other suitable hostcell can also be used for expressing-heterologous or endogenous nucleicacid encoding an antibody, specified fragment or variant thereof, of thepresent disclosure. The fused cells (hybridomas) or recombinant cellscan be isolated using selective culture conditions or other suitableknown methods, and cloned by limiting dilution or cell sorting, or otherknown methods.

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oligonucleotide, cDNA, or the like, display library; e.g., as availablefrom various commercial vendors such as MorphoSys (Martinsreid/Planegg,Del.), BioInvent (Lund, Sweden), Affitech (Oslo, Norway) using methodsknown in the art. Art known methods are described in the patentliterature some of which include U.S. Pat. Nos. 4,704,692; 5,723,323;5,763,192; 5,814,476; 5,817,483; 5,824,514; and 5,976,862. Alternativemethods rely upon immunization of transgenic animals (e.g., SCID mice,Nguyen et al. (1977) Microbiol. Immunol. 41:901-907 (1997); Sandhu etal. (1996) Crit, Rev. Biotechnol. 16:95-118; Eren et al. (1998) Mumma93:154-161 that are capable of producing a repertoire of humanantibodies, as known in the art and/or as described herein. Suchtechniques, include, but are not limited to, ribosome display Wanes etal. (1997) Proc. Natl. Acad. Sci. USA 94:4937-4942; Hanes et al. (1998)Proc. Natl. Acad. Sci. USA 95:14130-14135); single cell antibodyproducing technologies (e.g., selected lymphocyte antibody method(“SLAM”) (U.S. Pat. No. 5,627,052; Wen et al. (1987) J. Immunol17:887-892; Babcook et al. (1996) Proc. Natl. Acad. Sci. USA93:7843-7848); gel microdroplet and flow cytometry (Powell et al. (1990)Biotechnol. 8:333-337; One Cell Systems, (Cambridge, Mass.); Gray et al.(1995) J. Imm. Meth. 182:155-163; and Kenny et al. (1995) Bio. Technol.13:787-790); B-cell selection (Steenbakkers et al. (1994) Molec. Biol.Reports 19:125-134).

Antibody derivatives of the present disclosure can also be prepared bydelivering a polynucleotide encoding an antibody disclosed herein to asuitable host such as to provide transgenic animals or mammals, such asgoats, cows, horses, sheep, and the like, that produce such antibodiesin their milk. These methods are known in the art and are described forexample in U.S. Pat. Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992;5,994,616; 5,565,362; and 5,304,489.

The term “antibody derivative” includes post-translational modificationto linear polypeptide sequence of the antibody or fragment. For example,U.S. Pat. No. 6,602,684 B1 describes a method for the generation ofmodified glycol-forms of antibodies, including whole antibody molecules,antibody fragments, or fusion proteins that include a region equivalentto the Fc region of an immunoglobulin, having enhanced Fe-mediatedcellular toxicity, and glycoproteins so generated.

The antibodies disclosed herein also include derivatives that aremodified by the covalent attachment of any type of molecule to theantibody such that covalent attachment does not prevent the antibodyfrom generating an anti-idiotypic response. Antibody derivativesinclude, but are not limited to, antibodies that have been modified byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc.Additionally, the derivatives may contain one or more non-classicalamino acids.

Antibody derivatives also can be prepared by delivering a polynucleotidedisclosed herein to provide transgenic plants and cultured plant cells(e.g., but not limited to tobacco, maize, and duckweed) that producesuch antibodies, specified portions or variants in the plant parts or incells cultured therefrom. For example, Cramer et al. (1999) Curr. Top.Microbol. Immunol. 240:95-118 and references cited therein, describe theproduction of transgenic tobacco leaves expressing large amounts ofrecombinant proteins, e.g., using an inducible promoter. Transgenicmaize have been used to express mammalian proteins at commercialproduction levels, with biological activities equivalent to thoseproduced in other recombinant systems or purified from natural sources.See, e.g., Hood et al. (1999) Adv. Exp. Med. Biol. 464:127-147 andreferences cited therein. Antibody derivatives have also been producedin large amounts from transgenic plant seeds including antibodyfragments, such as single chain antibodies (scFv's), including tobaccoseeds and potato tubers. See, e.g., Conrad et al. (1998) Plant Mol.Biol. 38:101-109 and references cited therein. Thus, antibodies can alsobe produced using transgenic plants, according to know methods.

Antibody derivatives also can be produced, for example, by addingexogenous sequences to modify immunogenicity or reduce, enhance ormodify binding, affinity, on-rate, off-rate, avidity, specificity,half-life, or any other suitable characteristic. Generally part or allof the non-human or human CDR sequences are maintained while thenon-human sequences of the variable and constant regions are replacedwith human or other amino acids or variable or constant regions fromother isotypes.

In general, the CDR residues are directly and most substantiallyinvolved in influencing antigen binding. Humanization or engineering ofantibodies can be performed using any known method such as, but notlimited to, those described in U.S. Pat. Nos. 5,723,323; 5,976,862;5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886;5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089;5,225,539; and 4,816,567.

Chimeric, humanized or primatized antibodies of the present disclosurecan be prepared based on the sequence of a reference monoclonal antibodyprepared using standard molecular biology techniques. DNA encoding theheavy and light chain immunoglobulins can be obtained from the hybridomaof interest and engineered to contain non-reference (e.g., human)immunoglobulin sequences using standard molecular biology techniques.For example, to create a chimeric antibody, the murine variable regionscan be linked to human constant regions using methods known in the art(U.S. Pat. No. 4,816,567). To create a humanized antibody, the murineCDR regions can be inserted into a human framework using methods knownin the art (U.S. Pat. Nos. 5,225,539 and 5,530,101; 5,585,089;5,693,762; and 6,180,370). Similarly, to create a primatized antibodythe murine CDR regions can be inserted into a primate framework usingmethods known in the art (WO 93/02108 and WO 99/55369).

Techniques for making partially to fully human antibodies are known inthe art and any such techniques can be used. According to oneembodiment, fully human antibody sequences are made in a transgenicmouse which has been engineered to express human heavy and light chainantibody genes. Multiple strains of such transgenic mice have been madewhich can produce different classes of antibodies. B cells fromtransgenic mice which are producing a desirable antibody can be fused tomake hybridoma cell lines for continuous production of the desiredantibody. (See for example, Russel et al. (2000) Infection and ImmunityApril 2000:1820-1826; Gallo et al. (2000) European J. of Immun.30:534-540; Green (1999) J. of Immun. Methods 231:11-23; Yang et al.(1999A) J. of Leukocyte Biology 66:401-410; Yang (1999B) Cancer Research59(6):1236-1243; Jakobovits (1998) Advanced Drug Reviews 31:33-42; Greenand Jakobovits (1998) J. Exp. Med. 188(3):483-495; Jakobovits (1998)Exp. Opin. Invest. Drugs 7(4):607-614; Tsuda et al. (1997) Genomics42:413-421; Sherman-Gold (1997) Genetic Engineering News 17(14); Mendezet al. (1997) Nature Genetics 15:146-156; Jakobovits (1996) Weir'sHandbook of Experimental Immunology, The Integrated Immune System Vol.IV, 194.1-194.7; Jakobovits (1995) Current Opinion in Biotechnology6:561-566; Mendez et al. (1995) Genomics 26:294-307; Jakobovits (1994)Current Biology 4(8):761-763; Arbones et al. (1994) Immunity1(4):247-260; Jakobovits (1993) Nature 362(6417):255-258; Jakobovits etal. (1993) Proc. Natl. Acad. Sci. USA 90(6):2551-2555; and U.S. Pat. No.6,075,181.)

The antibodies disclosed herein also can be modified to create chimericantibodies. Chimeric antibodies are those in which the various domainsof the antibodies' heavy and light chains are coded for by DNA from morethan one species. See, e.g., U.S. Pat. No. 4,816,567.

Alternatively, the antibodies disclosed herein can also be modified tocreate veneered antibodies. Veneered antibodies are those in which theexterior amino acid residues of the antibody of one species arejudiciously replaced or “veneered” with those of a second species sothat the antibodies of the first species will not be immunogenic in thesecond species thereby reducing the immunogenicity of the antibody.Since the antigenicity of a protein is primarily dependent on the natureof its surface, the immunogenicity of an antibody could be reduced byreplacing the exposed residues which differ from those usually found inanother mammalian species antibodies. This judicious replacement ofexterior residues should have little, or no, effect on the interiordomains, or on the interdomain contacts. Thus, ligand binding propertiesshould be unaffected as a consequence of alterations which are limitedto the variable region framework residues. The process is referred to as“veneering” since only the outer surface or skin of the antibody isaltered, the supporting residues remain undisturbed.

The procedure for “veneering” makes use of the available sequence datafor human antibody variable domains compiled by Kabat et al. (1987)Sequences of Proteins of Immunological interest, 4th ed., Bethesda, Md.,National Institutes of Health, updates to this database, and otheraccessible U.S. and foreign databases (both nucleic acid and protein).Non-limiting examples of the methods used to generate veneeredantibodies include EP 519596; U.S. Pat. No. 6,797,492; and described inPadlan et al. (1991) Mol. Immunol. 28(4-5):489-498.

The term “antibody derivative” also includes “diabodies” which are smallantibody fragments with two antigen-binding sites, wherein fragmentscomprise a heavy chain variable domain (VH) connected to a light chainvariable domain (VL) in the same polypeptide chain. (See for example, EP404,097; WO 93/11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci.USA 90:6444-6448.) By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. (See also, U.S. Pat. No. 6,632,926 to Chen etal., which discloses antibody variants that have one or more amino acidsinserted into a hypervariable region of the parent antibody and abinding affinity for a target antigen which is at least about two foldstronger than the binding affinity of the parent antibody for theantigen).

The term “antibody derivative” further includes engineered antibodymolecules, fragments and single domains such as scFv, dAbs, nanobodies,minibodies, Unibodies, and Affibodies & Hudson (2005) Nature Biotech23(9):1126-36; U.S. Pat. Application Publication No. 2006/0211088; PCTInternational Application Publication No. WO 2007/059782; U.S. Pat. No.5,831,012).

The term “antibody derivative” further includes “linear antibodies”. Theprocedure for making linear antibodies is known in the art and describedin Zapata et al. (1995) Protein Eng. 8(10):1057-1062. Briefly, theseantibodies comprise a pair of tandem Ed segments(V_(H)-C_(H)1-VH-C_(H)1) which form a pair of antigen binding regions.Linear antibodies can be bispecific or monospecific.

The antibodies disclosed herein can be recovered and purified fromrecombinant cell cultures by known methods including, but not limitedto, protein A purification, ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe used for purification.

Antibodies of the present disclosure include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a eukaryotic host, including,for example, yeast, higher plant, insect and mammalian cells, oralternatively from a prokaryotic host as described above. A number ofantibody production systems are described in Birch & Radner (2006) Adv.Drug Delivery Rev. 58: 671-685.

If an antibody being tested binds with protein or polypeptide, then theantibody being tested and the antibodies provided by this disclosure areequivalent. It also is possible to determine without undueexperimentation, whether an antibody has the same specificity as theantibody disclosed herein by determining whether the antibody beingtested prevents an antibody disclosed herein from binding the protein orpolypeptide with which the antibody is normally reactive. If theantibody being tested competes with the antibody disclosed herein asshown by a decrease in binding by the monoclonal antibody disclosedherein, then it is likely that the two antibodies bind to the same or aclosely related epitope. Alternatively, one can pre-incubate theantibody disclosed herein with a protein with which it is normallyreactive, and determine if the antibody being tested is inhibited in itsability to bind the antigen. If the antibody being tested is inhibitedthen, in all likelihood, it has the same, or a closely related, epitopicspecificity as the antibody disclosed herein.

The term “antibody” also is intended to include antibodies of allimmunoglobulin isotypes and subclasses. Particular isotypes of amonoclonal antibody can be prepared either directly by selecting from aninitial fusion, or prepared secondarily, from a parental hybridomasecreting a monoclonal antibody of different isotype by using the sibselection technique to isolate class switch variants using the proceduredescribed in Steplewski et al. (1985) Proc. Natl. Acad. Sci. USA 82:8653or Spira et al. (1984) J. Immunol. Methods 74:307. Alternatively,recombinant DNA techniques may be used.

The isolation of other monoclonal antibodies with the specificity of themonoclonal antibodies described herein can also be accomplished by oneof ordinary skill in the art by producing anti-idiotypic antibodies.Herlyn et al. (1986) Science 232:100. An anti-idiotypic antibody is anantibody which recognizes unique determinants present on the monoclonalantibody of interest.

In some aspects disclosed herein, it will be useful to detectably ortherapeutically label the antibody. Suitable labels are described supra.Methods for conjugating antibodies to these agents are known in the art.For the purpose of illustration only, antibodies can be labeled with adetectable moiety such as a radioactive atom, a chromophore, afluorophore, or the like. Such labeled antibodies can be used fordiagnostic techniques, either in vivo, or in an isolated test sample.

The coupling of antibodies to low molecular weight haptens can increasethe sensitivity of the antibody in an assay. The haptens can then bespecifically detected by means of a second reaction. For example, it iscommon to use haptens such as biotin, which reacts avidin, ordinitrophenol, pyridoxal, and fluorescein, which can react with specificanti-hapten antibodies. See, Harlow and Lane (1988) supra.

The variable region of the antibodies of the present disclosure can bemodified by mutating amino acid residues within the VII and/or VL CDR 1,CDR 2 and/or CDR 3 regions to improve one or more binding properties(e.g., affinity) of the antibody. Mutations may be introduced bysite-directed mutagenesis or PCR-mediated mutagenesis and the effect onantibody binding, or other functional property of interest, can beevaluated in appropriate in vitro or in vivo assays. In certainembodiments, conservative modifications are introduced and typically nomore than one, two, three, four or five residues within a CDR region arealtered. The mutations may be amino acid substitutions, additions ordeletions.

Framework modifications can be made to the antibodies to decreaseimmunogenicity, for example, by “backmutating” one or more frameworkresidues to the corresponding germline sequence.

In addition, the antibodies disclosed herein may be engineered toinclude modifications within the Fc region to alter one or morefunctional properties of the antibody, such as serum half-fife,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Such modifications include, but are not limitedto, alterations of the number of cysteine residues in the hinge regionto facilitate assembly of the light and heavy chains or to increase ordecrease the stability of the antibody (U.S. Pat. No. 5,677,425) andamino acid mutations in the Fc hinge region to decrease the biologicalhalf-life of the antibody (U.S. Pat.

Additionally, the antibodies disclosed herein may be chemicallymodified. Glycosylation of an antibody can be altered, for example, bymodifying one or more sites of glycosylation within the antibodysequence to increase the affinity of the antibody for antigen (U.S. Pat.Nos. 5,714,350 and 6,350,861). Alternatively, to increaseantibody-dependent cell-mediated cytotoxicity, a hypofucosylatedantibody having reduced amounts of fucosyl residues or an antibodyhaving increased bisecting GlcNac structures can be obtained byexpressing the antibody in a host cell with altered glycosylationmechanism (Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740;Umana et al. (1999) Nat. Biotech. 17:176-180).

The antibodies disclosed herein can be pegylated to increase biologicalhalf-life by reacting the antibody or fragment thereof with polyethyleneglycol (PEG) or a reactive ester or aldehyde derivative of PEG, underconditions in which one or more PEG groups become attached to theantibody or antibody fragment. Antibody pegylation may be carried out byan acylation reaction or an alkylation reaction with a reactive PEGmolecule (or an analogous reactive water soluble polymer). As usedherein, the term “polyethylene glycol” is intended to encompass any ofthe forms of PEG that have been used to derivatize other proteins, suchas mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethyleneglycol-maleimide. The antibody to be pegylated can be an aglycosylatedantibody. Methods for pegylating proteins are known in the art and canbe applied to the antibodies disclosed herein (EP 0154316 and EP0401384).

Additionally, antibodies may be chemically modified by conjugating orfusing the antigen-binding region of the antibody to serum protein, suchas human serum albumin, to increase half-life of the resulting molecule.Such approach is for example described in EP 0322094 and EP 0486525.

The antibodies or fragments thereof of the present disclosure may beconjugated to a diagnostic agent and used diagnostically, for example,to monitor the development or progression of a disease and determine theefficacy of a given treatment regimen. Examples of diagnostic agentsinclude enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive materials, positronemitting metals using various positron emission tomographies, andnonradioactive paramagnetic metal ions. The detectable substance may becoupled or conjugated either directly to the antibody or fragmentthereof, or indirectly, through a linker using techniques known in theart. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase.Examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin. Examples of suitable fluorescentmaterials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin. An example of a luminescent material includesluminol. Examples of bioluminescent materials include luciferase,luciferin, and aequorin. Examples of suitable radioactive materialinclude ¹²⁵I,¹³¹I, Indium-111, Lutetium-171, Bismuth-212, Bismuth-213,Astatine-211, Copper-62, Copper-64, Copper-67, Yttrium-90, Iodine-125,Iodine-131, Phosphorus-32, Phosphorus-33, Scandium-47, Silver-111,Gallium-67, Praseodymium-142, Samarium-153, Terbium-161, Dysprosium-166,Holmium-166, Rhenium-186, Rhenium-188, Rhenium-189, Lead-212,Radium-223, Actinium-225, Iron-59, Selenium-75, Arsenic-77,Strontium-89, Molybdenum-99, Rhodium-1105, Palladium-109,Praseodymium-143, Promethium-149, Erbium-169, Iridium-194, Gold-198,Gold-199, and Lead-211. Monoclonal antibodies may be indirectlyconjugated with radiometal ions through the use of bifunctionalchelating agents that are covalently linked to the antibodies. Chelatingagents may be attached through amities (Meares et al. (1984) Anal.Biochem. 142:68-78); sulfhydryl groups (Koyama (1994) Chem. Abstr.120:217-262) of amino acid residues and carbohydrate groups (Rodwell etal. (1986) PNAS USA 83:2632-2636; Quadri et al. (1993) Nucl. Med. Biol.20:559-570).

Further, the antibodies or fragments thereof of the present disclosuremay be conjugated to a therapeutic agent. Suitable therapeutic agentsinclude taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin,antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, fludarabin, 5-fluorouracil, decarbazine, hydroxyurea,asparaginase, gemcitabinc, cladribine), alkylating agents (such asmechlorethamine, thioepa, chloramhucil, melphalan, carmustine (BSNU),lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatinand other platinum derivatives, such as carboplatin), antibiotics (suchas dactinomycin (formerly actinomycin), bleomycin, daunorubicin(formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin,mitoxantrone, plicamycin, anthramycin (AMC)), diphtheria toxin andrelated molecules (such as diphtheria A chain and active fragmentsthereof and hybrid molecules), ricin toxin (such as ricin A or adeglycosylated ricin A chain toxin), cholera toxin, a Shiga-like toxin(SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussistoxin, tetanus toxin, soybean Bowman-Birk protease inhibitor,Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain,modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthinproteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S),Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalisinhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycintoxins and mixed toxins.

Additional suitable conjugated molecules include ribonuclease (RNase),DNase I, an antisense nucleic acid, an inhibitory RNA molecule such as asiRNA molecule, an immunostimulatory nucleic acid, aptamers, ribozymes,triplex forming molecules, and external guide sequences. Aptamers aresmall nucleic acids ranging from 15-50 bases in length that fold intodefined secondary and tertiary structures, such as stem-loops orG-quartets, and can bind small molecules, such as ATP (U.S. Pat. No.5,631,146) and theophiline (U.S. Pat. No. 5,580,737), as well as largemolecules, such as reverse transcriptase (U.S. Pat. No. 5,786,462) andthrombin (U.S. Pat. No. 5,543,293). Ribozymes are nucleic acid moleculesthat are capable of catalyzing a chemical reaction, eitherintramolecularly or intermolecularly. Ribozymes typically cleave nucleicacid substrates through recognition and binding of the target substratewith subsequent cleavage. Triplex forming function nucleic acidmolecules can interact with double-stranded or single-stranded nucleicacid by forming a triplex, in which three strands of DNA form a complexdependent on both Watson-Crick and Hoogsteen base-pairing. Triplexmolecules can bind target regions with high affinity and specificity.

The functional nucleic acid molecules may act as effectors, inhibitors,modulators, and stimulators of a specific activity possessed by a targetmolecule, or the functional nucleic acid molecules may possess a de novoactivity independent of any other molecules.

The therapeutic agents can be linked to the antibody directly orindirectly, using any of a large number of available methods. Forexample, an agent can be attached at the hinge region of the reducedantibody component via disulfide bond formation, using cross-linkerssuch as N-succinyl 3-(2-pyridyldithio)proprionate (SPDP), or via acarbohydrate moiety in the Fc region of the antibody (Yu et al. 1994Int. J. Cancer 56: 244; Upeslacis et al., “Modification of Antibodies byChemical Methods,” in Monoclonal antibodies: principles andapplications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal antibodies: Production,engineering and clinical application, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995)).

Techniques for conjugating therapeutic agents to antibodies are wellknown (Amon et al. “Monoclonal Antibodies For Immunotargeting Of DrugsIn Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy;Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstromet al. “Antibodies For Drug Delivery,” in Controlled Drug Delivery (2ndEd.); Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987);Thorpe “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: AReview,” in Monoclonal Antibodies '84: Biological And ClinicalApplications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis,Results, And Future Prospective Of The Therapeutic Use Of RadiolabeledAntibody in Cancer Therapy,” in Monoclonal Antibodies For CancerDetection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press1985), and Thorpe et al. “The Preparation And Cytotoxic Properties OfAntibody-Toxin Conjugates,” (1982) Immunol. Rev. 62:119-58).

The antibodies disclosed herein or antigen-binding regions thereof canbe linked to another functional molecule such as another antibody orligand for a receptor to generate a bi-specific or multi-specificmolecule that binds to at least two or more different binding sites ortarget molecules. Linking of the antibody to one or more other bindingmolecules, such as another antibody, antibody fragment, peptide orbinding mimetic, can be done, for example, by chemical coupling, geneticfusion, or noncovalent association. Multi-specific molecules can furtherinclude a third binding specificity, in addition to the first and secondtarget epitope.

Bi-specific and multi-specific molecules can be prepared using methodsknown in the art. For example, each binding unit of the hi-specificmolecule can be generated separately and then conjugated to one another.When the binding molecules are proteins or peptides, a variety ofcoupling or cross-linking agents can be used for covalent conjugation.Examples of cross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitroberizoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate(sulfo-SMCC) (Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu et al.(1985) Proc. Natl. Acad. Sci. USA 82:8648). When the binding moleculesare antibodies, they can be conjugated by sulfhydryl bonding of theC-terminus hinge regions of the two heavy chains.

The antibodies or fragments thereof of the present disclosure may belinked to a moiety that is toxic to a cell to which the antibody isbound to form “depleting” antibodies. These antibodies are particularlyuseful in applications where it is desired to deplete an NK cell.

The antibodies disclosed herein may also be attached to solid supports,which are particularly useful for immunoassays or purification of thetarget antigen. Such solid supports include, but are not limited to,glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chlorideor polypropylene.

The antibodies also can be bound to many different carriers. Thus, thisdisclosure also provides compositions containing the antibodies andanother substance, active or inert. Examples of well-known carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylase, natural and modified cellulose, polyacrylamide, agarose, andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes disclosed herein. Those skilled in the art will know ofother suitable carriers for binding monoclonal antibodies, or will beable to ascertain such, using routine experimentation.

In certain aspects, the disclosure relates to an antibody or antigenbinding fragment that specifically recognizes or binds an isolated orrecombinant polypeptide consisting essentially of an amino acid sequenceof a DNABII protein or fragment thereof, the tail fragment or tipfragment. Non-limiting examples of such fragments include withoutlimitation a polypeptide that comprises one or more of the sequences:polypeptides designated as B1 through B6 (see FIG. 18 );MATITKLDIIEYLSDKYHLS (also referred to herein as hIFA1; (SEQ ID NO.348)); KYHLSKQDTKNVVENFLEEI (also referred to herein as hIFA2; (SEQ IDNO. 349)); FLEEIRLSLESGQDVKLSGF (also referred to herein as hIFA3; (SEQID NO. 350)); KLSGFGNFELRDKSSRPGRN (also referred to herein as hIFA4;(SEQ ID NO. 351)); RPGRNPKTGDVVPVSARRVV (also referred to herein ashIFA5; (SEQ ID NO. 352)); ARRVVTFKPGQKLRARVEKTK (also referred to hereinas hIFA6; (SEQ ID NO. 353)); or an equivalent of each thereof, that mayinclude additional amino acids as described above;

Non-limiting exemplary antibodies produced by the disclosed hybridomasdisclosed in Table 10. The hybridoma cell lines that produce monoclonalantibodies that specifically recognize and bind Haemophilus influenzaeIHfA fragment A5 (SEQ ID NO: 352) and Ihf fragment B4 (SEQ ID NO: 345),were deposited with American Type Culture Collection (ATCC) pursuant tothe provisions of the Budapest Treaty on Jul. 30, 2015 and the CDRs ofthe antibodies can be identified by sequence using conventionaltechniques. Further non-limiting exemplary antibodies include those thatspecifically recognize and bind Haemophilus influenzae IHfA fragment A3(SEQ ID NO: 350), IhfB fragment B2 (SEQ ID NO: 343) produced byhybridoma cell lines IhfA3 NTHI 9B10.F2.H3 and IhfB2 NTHI 7A4.E4.G4.

TABLE 10 ATCC Specificity SEQ ID NO: Accession No. Hybridoma Cell LineIhfA frag. A5 SEQ ID NO: PTA-122334 IhfA5 NTHI 14G8.F5.G6 352 IhfB frag.B4 SEQ ID NO: PTA-122335 IhfB4 NTHI 4E11.E5.G2 345

In one aspect, the present disclosure provides an isolated antibody,derivative or fragment thereof that is at least 85% identical to anantibody selected from the group consisting of (i) the antibody producedby hybridoma cell line IhfA5 NTHI 14G8.F5.G6 or (ii) the antibodyproduced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2.

In one aspect, the present disclosure provides an isolated antibody,derivative or fragment thereof comprising the CDRs of (i) the antibodyproduced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6; or (ii) theantibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2. In oneaspect, the present disclosure provides an isolated antibody, derivativeor fragment thereof that has CDR that is at least 85% identical to (i)the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6 or(ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2.

In some aspects of the antibodies provided herein, the HC variabledomain sequence comprises the HC variable domain sequence of (i) theantibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6 or (ii)the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2,and/or the LC variable domain sequence comprises the LC variable domainsequence of (i) the antibody produced by hybridoma cell line IhfA5 NTHI14G8.F5.G6 or (ii) the antibody produced by hybridoma cell line IhfB4NTHI 4E11.E5.G2.

In some aspects of the antibodies provided herein, the HC variabledomain sequence comprises a HC variable domain sequence at least 85%identical to a HC variable domain sequence of (i) the antibody producedby hybridoma cell line IhfA5 NTHI 14G8.F5.G6; or (ii) the antibodyproduced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2; and/or the LCvariable domain sequence comprises a LC variable domain sequence atleast 85% identical to the of LC variable domain sequence of (i) theantibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6; or (ii)the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2.

In one aspect, the present disclosure provides an isolated antibodycomprising a heavy chain (HC) variable domain sequence and a light chain(LC) variable domain sequence, wherein the heavy chain and light chainimmunoglobulin variable domain sequences form an antigen binding sitethat binds to an epitope of a DNABII protein.

In some embodiments, the heavy chain variable region comprises a CDRH1sequence comprising, or alternatively consisting essentially of, or yetfurther consisting of, an amino acid sequence comprising the CDRH1 ofany one of the following antibodies: (i) the antibody produced byhybridoma cell line IhfA5 NTHI 14G8.F5.G6; or (ii) the antibody producedby hybridoma cell line IhfB4 NTHI 4E11.E5.G2.

In some embodiments, the heavy chain variable region comprises a CDRH2sequence comprising, or alternatively consisting essentially of, or yetfurther consisting of, an amino acid sequence comprising the CDRH2 ofany one of the following antibodies: (i) the antibody produced byhybridoma cell line IhfA5 NTHI 14G8.F5.G6; or (ii) the antibody producedby hybridoma cell line IhfB4 NTHI 4E11.E5.G2.

In some embodiments, the heavy chain variable region comprises a CDRH3sequence comprising, or alternatively consisting essentially of, or yetfurther consisting of, an amino acid sequence comprising the CDRH3 ofany one of the following antibodies: (i) the antibody produced byhybridoma cell line IhfA5 NTHI 14G8.F5.G6; or (ii) the antibody producedby hybridoma cell line IhfB4 NTHI 4E11.E5.G2.

In some embodiments, the heavy chain variable region comprises, oralternatively consists essentially of, or yet further consists of, theamino acid sequence comprising the heavy chain variable region sequenceof any one of the following antibodies: (i) the antibody produced byhybridoma cell line IhfA5 NTHI 14G8.F5.G6; or (ii) the antibody producedby hybridoma cell line IhfB4 NTHI 4E11.E5.G2.

In some embodiments, the light chain variable region comprises a CDRL1sequence comprising, or alternatively consisting essentially of, or yetfurther consisting of, an amino acid sequence comprising the CDRL1 ofany one of the following antibodies: (i) the antibody produced byhybridoma cell line IhfA5 NTHI 14G8.F5.G6; or (ii) the antibody producedby hybridoma cell line IhfB4 NTHI 4E11.E5.G2.

In some embodiments, the light chain variable region comprises a CDRL2sequence comprising, or alternatively consisting essentially of, or yetfurther consisting of, an amino acid sequence comprising the CDRL2 ofany one of the following antibodies: (i) the antibody produced byhybridoma cell line IhfA5 NTHI 14G8.F5.G6; or (ii) the antibody producedby hybridoma cell line IhfB4 NTHI 4E11.E5.G2.

In some embodiments, the light chain variable region comprises a CDRL3sequence comprising, or alternatively consisting essentially of, or yetfurther consisting of, an amino acid sequence comprising the CDRL3 ofany one of the following antibodies: (i) the antibody produced byhybridoma cell line IhfA5 NTHI 14G8.F5.G6; or (ii) the antibody producedby hybridoma cell line IhfB4 NTHI 4E11.E5.G2.

In some embodiments, the light chain variable region comprises, oralternatively consists essentially of, or yet further consists of, thepolypeptide encoded by the polynucleotide sequence comprising the lightchain variable region sequence of any one of the following antibodies:(i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6;or (ii) the antibody produced by hybridoma cell line IhfB4 NTHI4E11.E5.G2.

In another aspect of the present technology, the isolated antibodyincludes one or more of the following characteristics:

(a) the light chain immunoglobulin variable domain sequence comprisesone or more CDRs that are at least 85% identical to a CDR of a lightchain variable domain of any of the disclosed light chain sequences;

(b) the heavy chain immunoglobulin variable domain sequence comprisesone or more CDRs that are at least 85% identical to a CDR of a heavychain variable domain of any of the disclosed heavy chain sequences;

(c) the light chain immunoglobulin variable domain sequence is at least85% identical to a light chain variable domain of any of the disclosedlight chain sequences;

(d) the HC immunoglobulin variable domain sequence is at least 85%identical to a heavy chain variable domain of any of the disclosed lightchain sequences; and/or

(e) the antibody binds an epitope that overlaps with an epitope bound byany of the disclosed sequences.

In some of the aspects of the antibodies provided herein, the antibodybinds a DNABII protein with a dissociation constant (K_(D)) of less than10⁻⁴M, 10⁻⁵M, 10⁻⁶ M, 10⁻⁷M, 10⁻⁸ M, 10⁻⁹M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹²M. In some of the aspects of the antibodies provided herein, the antigenbinding site specifically binds to a DNABII protein.

In some of the aspects of the antibodies provided herein, the antibodyis soluble Fab.

In some of the aspects of the antibodies provided herein, the HC and LCvariable domain sequences are components of the same polypeptide chain.In some of the aspects of the antibodies provided herein, the HC and LCvariable domain sequences are components of different polypeptidechains.

In some of the aspects of the antibodies provided herein, the antibodyis a full-length antibody.

In some of the aspects of the antibodies provided herein, the antibodyis a monoclonal antibody.

In some of the aspects of the antibodies provided herein, the antibodyis chimeric or humanized.

In some of the aspects of the antibodies provided herein, the antibodyis selected from the group consisting of Fab, F(ab)′2, Fab′, scFv, andFv.

In some of the aspects of the antibodies provided herein, the antibodycomprises an Fc domain. In some of the aspects of the antibodiesprovided herein, the antibody is a non-human animal such as a rat,sheep, bovine, canine, feline or rabbit antibody. In some of the aspectsof the antibodies provided herein, the antibody is a human or humanizedantibody or is non-immunogenic in a human.

In some of the aspects of the antibodies provided herein, the antibodycomprises a human antibody framework region.

In other aspects, one or more amino acid residues in a CDR of theantibodies provided herein are substituted with another amino acid. Thesubstitution may be “conservative” in the sense of being a substitutionwithin the same family of amino acids. The naturally occurring aminoacids may be divided into the following four families and conservativesubstitutions will take place within those families.

1) Amino acids with basic side chains: lysine, arginine, histidine.

2) Amino acids with acidic side chains: aspartic acid, glutamic acid

3) Amino acids with uncharged polar side chains: asparagine, glutamine,serine, threonine, tyrosine.

4) Amino acids with nonpolar side chains: glycine, alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan,cysteine.

In another aspect, one or more amino acid residues are added to ordeleted from one or more CDRs of an antibody. Such additions ordeletions occur at the N or C termini of the CDR or at a position withinthe CDR.

By varying the amino acid sequence of the CDRs of an antibody byaddition, deletion or substitution of amino acids, various effects suchas increased binding affinity for the target antigen may be obtained.

It is to be appreciated that antibodies of the present disclosurecomprising such varied CDR sequences still bind a DNABII protein withsimilar specificity and sensitivity profiles as the disclosedantibodies. This may be tested by way of the binding assays.

In a further aspect, the antibodies are characterized by being bothimmunodominant and immunoprotective, as determined using appropriateassays and screens.

Isolation, Culturing and Expansion of APCs, Including Dendritic Cells

This disclosure also provides isolated host cells comprising one or moreof an isolated polypeptides or isolated polynucleotides or the vectorsdisclosed herein. In one aspect the isolated host cell is a eukaryoticcell such as antigen presenting cell (APC), e.g., a dendritic cell. Inanother aspect, the isolated host cell is a prokaryotic cell. In oneaspect, the disclosure is an isolated host cell that is cultured underconditions that promote expression of the polynucleotide. Thisdisclosure also provides the host cell, expression system andpolypeptide produced by the expression system.

The following is a brief description of two fundamental approaches forthe isolation of APC. These approaches involve (1) isolating bone marrowprecursor cells (CD34⁺) from blood and stimulating them to differentiateinto APC; or (2) collecting the precommitted APCs from peripheral blood.In the first approach, the patient must be treated with cytokines suchas GM-CSF to boost the number of circulating CD34⁺ stem cells in theperipheral blood.

The second approach for isolating APCs is to collect the relativelylarge numbers of precommitted APCs already circulating in the blood.Previous techniques for isolating committed APCs from human peripheralblood have involved combinations of physical procedures such asmetrizamide gradients and adherence/nonadherence steps (Freudenthal etal. (1990) PNAS 87:7698-7702); Percoll gradient separations(Mehta-Damani et al. (1994) J. Immunol. 153:996-1003); and fluorescenceactivated cell sorting techniques (Thomas et al. (1993) J. Immunol.151:6840-6852).

One technique for separating large numbers of cells from one another isknown as countercurrent centrifugal elutriation (CCE). Cell samples areplaced in a special elutriation rotor. The rotor is then spun at aconstant speed of, for example, 3000 rpm. Once the rotor has reached thedesired speed, pressurized air is used to control the flow rate ofcells. Cells in the elutriator are subjected to simultaneouscentrifugation and a washout stream of buffer which is constantlyincreasing in flow rate. This results in fractional cell separationsbased largely but not exclusively on differences in cell size.

In one aspect disclosed herein, the APC are precommitted or maturedendritic cells which can be isolated from the white blood cell fractionof a mammal, such as a murine, simian or a human (See, e.g., WO96/23060). The white blood cell fraction can be from the peripheralblood of the mammal. This method includes the following steps: (a)providing a white blood cell fraction obtained from a mammalian sourceby methods known in the art such as leukapheresis; (b) separating thewhite blood cell fraction of step (a) into four or more subfractions bycountercurrent centrifugal elutriation, (c) stimulating conversion ofmonocytes in one or more fractions from step (b) to dendritic cells bycontacting the cells with calcium ionophore, GM-CSF and IL-13 or GM-CSFand IL-4, (d) identifying the dendritic cell-enriched fraction from step(c), and (e) collecting the enriched fraction of step (d), optionally atabout 4° C. One way to identify the dendritic cell-enriched fraction isby fluorescence-activated cell sorting. The white blood cell fractioncan be treated with calcium ionophore in the presence of othercytokines, such as recombinant (rh) rhIL-12, rhGM-CSF, or rhIL-4. Thecells of the white blood cell fraction can be washed in buffer andsuspended in Ca⁺⁺/Mg⁺⁺ free media prior to the separating step. Thewhite blood cell fraction can be obtained by leukapheresis. Thedendritic cells can be identified by the presence of at least one of thefollowing markers: HLA-DR, HLA-DQ, or B7.2, and the simultaneous absenceof the following markers: CD3, CD16, CD56, CD57, and CD19, CD20.Monoclonal antibodies specific to these cell surface markers arecommercially available.

More specifically, the method requires collecting an enriched collectionof white cells and platelets from leukapheresis that is then furtherfractionated by countercurrent centrifugal elutriation (CCE) (Abrahamsenet al. (1991) J. Clin. Apheresis. 6:48-53). In this technique, cells aresubject to simultaneous centrifugation and a washout stream of bufferwhich is constantly increasing flow rate. The constantly increasingcountercurrent flow of buffer leads to fractional cell separations thatare largely based on cell size.

Quality control of APC and more specifically DC collection andconfirmation of their successful activation in culture is dependent upona simultaneous multi-color FACS analysis technique which monitors bothmonocytes and the dendritic cell subpopulation as well as possiblecontaminant T lymphocytes. Cell sorting is based on differentialexpression of cell surface markers including CD3 (T cells),CD16/CD56/CD57 (NK/LAK cells), and CD19/CD20 (B cells). DCs aredistinguishable from monocytes based in part on levels of CD14, which isexpressed at very high levels in monocytes compared to DCs. DCs showhigh levels of expression of HLA-DR, significant HLA-DQ and B7.2 (butlittle or no B7.1) at the time they are circulating in the blood (inaddition they express Leu M7 and M9, myeloid markers which are alsoexpressed by monocytes and neutrophils).

When combined with a third color reagent for analysis of dead cells,propidium iodide (PI), it is possible to make positive identification ofall cell subpopulations

The goal of FACS analysis at the time of collection is to confirm thatthe DCs are enriched in the expected fractions, to monitor neutrophilcontamination, and to make sure that appropriate markers are expressed.This rapid bulk collection of enriched DCs from human peripheral blood,suitable for clinical applications, is absolutely dependent on theanalytic FACS technique described above for quality control. If need be,mature DCs can be immediately separated from monocytes at this point byfluorescent sorting for “cocktail negative” cells. It may not benecessary to routinely separate DCs from monocytes because, themonocytes themselves are still capable of differentiating into DCs orfunctional DC-like cells in culture.

Once collected, the DC rich/monocyte APC fractions (usually 150 through190) can be pooled and cryopreserved for future use, or immediatelyplaced in short term culture.

Alternatively, others have reported that a method for upregulating(activating) dendritic cells and converting monocytes to an activateddendritic phenotype. This method involves the addition of calciumionophore to the culture media convert monocytes into activateddendritic cells. Adding the calcium ionophore A23187, for example, atthe beginning of a 24-48 hour culture period resulted in uniformactivation and dendritic cell phenotypic conversion of the pooled“monocyte plus DC” fractions: characteristically, the activatedpopulation becomes uniformly CD14 (Leu M3) negative, and upregulatesHLA-DR, HLA-DQ, ICAM-1, B7.1, and B7.2. Furthermore this activated bulkpopulation functions as well on a small numbers basis as a furtherpurified.

Specific combination(s) of cytokines have been used successfully toamplify (or partially substitute) for the activation/conversion achievedwith calcium ionophore: these cytokines include but are not limited topurified or recombinant (“rh”) rhGM-CSF, rhIL-2, and rhIL-4. Eachcytokine when given alone is inadequate for optimal upregulation.

Presentation of Antigen to the APC

For purposes of immunization, the polypeptides (e.g., SEQ ID NO: 1through 33) can be delivered to antigen-presenting cells asprotein/peptide or in the form of cDNA encoding the protein/peptide.Antigen-presenting cells (APCs) can consist of dendritic cells (DCs),monocytes/macrophages, B lymphocytes or other cell type(s) expressingthe necessary MHC/co-stimulatory molecules. The methods described belowfocus primarily on DCs which are the most potent APCs.

Pulsing is accomplished in vitro/ex vivo by exposing APCs to theantigenic protein or polypeptide(s) disclosed herein. The protein orpeptide(s) are added to APCs at a concentration of 1-10 μm forapproximately 3 hours. Transfection of APCs with polynucleotidesencoding antigens or antigenic polypeptides is accomplished by exposingAPCs to the nucleic acids in the presence of transfection agents knownin the art, including but not limited to cationic lipids. Transfected orpulsed APCs can subsequently be administered to the host via anintravenous, subcutaneous, intranasal, intramuscular or intraperitonealroute of delivery.

Protein/peptide antigen can also be delivered in vivo with adjuvant viathe intravenous, subcutaneous, intranasal, intramuscular orintraperitoneal route of delivery.

Foster Antigen Presenting Cells

Foster antigen presenting cells are particularly useful as a targetcell. Foster APCs are derived from the human cell line 174X CEM.T2,referred to as T2, which contains a mutation in its antigen processingpathway that restricts the association of endogenous peptides with cellsurface MHC class I molecules (Zweerink et al. (1993) J. Immunol.150:1763-1771). This is due to a large homozygous deletion in the MHCclass II region encompassing the genes TAP 1, TAP2, LMP 1, and LMP2,which are required for antigen presentation to MHC class 1-restrictedCD8⁺ CTLs. In effect, only “empty” MHC class I molecules are presentedon the surface of these cells. Exogenous peptide added to the culturemedium binds to these MHC molecules provided that the peptide containsthe allele-specific binding motif. These T2 cells are referred to hereinas “foster” APCs. They can be used in conjunction with this disclosureto present antigen(s).

Transduction of T2 cells with specific recombinant MHC allows forredirection of the MHC restriction profile. Libraries tailored to therecombinant allele will be preferentially presented by them because theanchor residues will prevent efficient binding to the endogenous allele.

High level expression of MHC molecules makes the APC more visible to theCTLs. Expressing the MHC allele of interest in T2 cells using a powerfultranscriptional promoter (e.g., the CMV promoter) results in a morereactive APC (most likely due to a higher concentration of reactiveMHC-peptide complexes on the cell surface).

Expansion of Immune Effector Cells

The present disclosure makes use of these APCs to stimulate productionof an enriched population of antigen-specific immune effector cells. Theantigen-specific immune effector cells are expanded at the expense ofthe APCs, which die in the culture. The process by which naive immuneeffector cells become educated by other cells is described essentiallyin Coulie (1997) Molec. Med. Today 3:261-268.

The APCs prepared as described above are mixed with naive immuneeffector cells. In specific embodiments, the cells may be cultured inthe presence of a cytokine, for example IL2. Because dendritic cellssecrete potent immunostimulatory cytokines, such as IL12, it may not benecessary to add supplemental cytokines during the first and successiverounds of expansion. In any event, the culture conditions are such thatthe antigen-specific immune effector cells expand (i.e., proliferate) ata much higher rate than the APCs. Multiple infusions of APCs andoptional cytokines can be performed to further expand the population ofantigen-specific cells.

In one embodiment, the immune effector cells are T cells. In a separateembodiment, the immune effector cells can be genetically modified bytransduction with a transgene coding for example, IL-2, IL-11 or IL-13.Methods for introducing transgenes in vitro, ex vivo and in vivo areknown in the art.

Functional Analysis with Antibodies

Antibodies disclosed herein can be used to purify the polypeptidesdisclosed herein and to identify biological equivalent polypeptideand/or polynucleotides. They also can be used to identify agents thatmodify the function of the polypeptides disclosed herein. Theseantibodies include polyclonal antisera, monoclonal antibodies, andvarious reagents derived from these preparations that are familiar tothose practiced in the art and described above.

Antibodies that neutralize the activities of proteins encoded byidentified genes can also be used in vivo and in vitro to demonstratefunction by adding such neutralizing antibodies into in vivo and invitro test systems. They also are useful as pharmaceutical agents tomodulate the activity of polypeptides disclosed herein.

Various antibody preparations can also be used in analytical methodssuch as ELISA assays or Western blots to demonstrate the expression ofproteins encoded by the identified genes by test cells in vitro or invivo. Fragments of such proteins generated by protease degradationduring metabolism can also be identified by using appropriate polyclonalantisera with samples derived from experimental samples.

The antibodies disclosed herein may be used for vaccination or to boostvaccination, alone or in combination with peptides or protein-basedvaccines or dendritic-cell based vaccines.

Compositions

Compositions are further provided. The compositions comprise a carrierand one or more of an isolated polypeptide disclosed herein, an isolatedpolynucleotide disclosed herein, a vector disclosed herein, an isolatedhost cell disclosed herein, a small molecule or an antibody disclosedherein. The carriers can be one or more of a solid support or apharmaceutically acceptable carrier. The compositions can furthercomprise an adjuvant or other components suitable for administrations asvaccines. In one aspect, the compositions are formulated with one ormore pharmaceutically acceptable excipients, diluents, carriers and/oradjuvants. In addition, embodiments of the compositions of the presentdisclosure include one or more of an isolated polypeptide disclosedherein, an isolated polynucleotide disclosed herein, a vector disclosedherein, a small molecule, an isolated host cell disclosed herein, or anantibody of the disclosure, formulated with one or more pharmaceuticallyacceptable substances.

For oral preparations, any one or more of an isolated or recombinantpolypeptide as described herein, an isolated or recombinantpolynucleotide as described herein, a vector as described herein, anisolated host cell as described herein, a small molecule or an antibodyas described herein can be used alone or in pharmaceutical formulationsdisclosed herein comprising, or consisting essentially of, the compoundin combination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents. Pharmaceuticallycompatible binding agents, and/or adjuvant materials can be included aspart of the composition. The tablets, pills, capsules, troches and thelike can contain any of the following ingredients, or compounds of asimilar nature: a binder such as microcrystalline cellulose, gumtragacanth or gelatin; an excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel, or corn starch; alubricant such as magnesium stearate or Sterotes; a glidant such ascolloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or orange flavoring.

Pharmaceutical formulations and unit dose forms suitable for oraladministration are particularly useful in the treatment of chronicconditions, infections, and therapies in which the patientself-administers the drug. In one aspect, the formulation is specificfor pediatric administration.

The disclosure provides pharmaceutical formulations in which the one ormore of an isolated polypeptide disclosed herein, an isolatedpolynucleotide disclosed herein, a vector disclosed herein, an isolatedhost cell disclosed herein, or an antibody disclosed herein can beformulated into preparations for injection in accordance with thedisclosure by dissolving, suspending or emulsifying them in an aqueousor nonaqueous solvent, such as vegetable or other similar oils,synthetic aliphatic acid glycerides, esters of higher aliphatic acids orpropylene glycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives or other antimicrobial agents. Anon-limiting example of such is a antimicrobial agent such as othervaccine components such as surface antigens, e.g., an OMP P5, OMP 26,OMP P2, or Type IV Pilin protein (see Jurcisek and Bakaletz (2007) J. ofBacteriology 189(10):3868-3875 and Murphy, T F, Bakaletz, L O andSmeesters, P R (2009) The Pediatric Infectious Disease Journal,28:S121-S126) and antibacterial agents. For intravenous administration,suitable carriers include physiological bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In allcases, a composition for parenteral administration must be sterile andshould be fluid to the extent that easy syringability exists.

Aerosol formulations provided by the disclosure can be administered viainhalation and can be propellant or non-propellant based. For example,embodiments of the pharmaceutical formulations disclosed herein comprisea compound disclosed herein formulated into pressurized acceptablepropellants such as dichlorodifluoromethane, propane, nitrogen and thelike. For administration by inhalation, the compounds can be deliveredin the form of an aerosol spray from a pressurized container ordispenser which contains a suitable propellant, e.g., a gas such ascarbon dioxide, or a nebulizer. A non-limiting example of anon-propellant is a pump spray that is ejected from a closed containerby means of mechanical force (i.e., pushing down a piston with one'sfinger or by compression of the container, such as by a compressiveforce applied to the container wall or an elastic force exerted by thewall itself, e.g., by an elastic bladder).

Suppositories disclosed herein can be prepared by mixing a compounddisclosed herein with any of a variety of bases such as emulsifyingbases or water-soluble bases. Embodiments of this pharmaceuticalformulation of a compound disclosed herein can be administered rectallyvia a suppository. The suppository can include vehicles such as cocoabutter, carbowaxes and polyethylene glycols, which melt at bodytemperature, yet are solidified at room temperature.

Unit dosage forms for oral or rectal administration, such as syrups,elixirs, and suspensions, may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more compoundsdisclosed herein. Similarly, unit dosage forms for injection orintravenous administration may comprise a compound disclosed herein in acomposition as a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier.

Embodiments of the pharmaceutical formulations disclosed herein includethose in which one or more of an isolated polypeptide disclosed herein,an isolated polynucleotide disclosed herein, a vector disclosed herein,a small molecule for use in the disclosure, an isolated host celldisclosed herein, or an antibody disclosed herein is formulated in aninjectable composition. Injectable pharmaceutical formulations disclosedherein are prepared as liquid solutions or suspensions; or as solidforms suitable for solution in, or suspension in, liquid vehicles priorto injection. The preparation may also be emulsified or the activeingredient encapsulated in liposome vehicles in accordance with otherembodiments of the pharmaceutical formulations disclosed herein.

In an embodiment, one or more of an isolated polypeptide disclosedherein, an isolated polynucleotide disclosed herein, a vector disclosedherein, an isolated host cell disclosed herein, or an antibody disclosedherein is formulated for delivery by a continuous delivery system. Theterm “continuous delivery system” is used interchangeably herein with“controlled delivery system” and encompasses continuous (e.g.,controlled) delivery devices (e.g., pumps) in combination withcatheters, injection devices, and the like, a wide variety of which areknown in the art.

Mechanical or electromechanical infusion pumps can also be suitable foruse with the present disclosure. Examples of such devices include thosedescribed in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019;4,487,603; 4,360,019; 4,725,852; 5,820,589; 5,643,207; 6,198,966; andthe like. In general, delivery of a compound disclosed herein can beaccomplished using any of a variety of refillable, pump systems. Pumpsprovide consistent, controlled release over time. In some embodiments, acompound disclosed herein is in a liquid formulation in adrug-impermeable reservoir, and is delivered in a continuous fashion tothe individual.

In one embodiment, the drug delivery system is an at least partiallyimplantable device. The implantable device can be implanted at anysuitable implantation site using methods and devices well known in theart. An implantation site is a site within the body of a subject atwhich a drug delivery device is introduced and positioned. Implantationsites include, but are not necessarily limited to, a subdermal,subcutaneous, intramuscular, or other suitable site within a subject'sbody. Subcutaneous implantation sites are used in some embodimentsbecause of convenience in implantation and removal of the drug deliverydevice.

Drug release devices suitable for use in the disclosure may be based onany of a variety of modes of operation. For example, the drug releasedevice can be based upon a diffusive system, a convective system, or anerodible system (e.g., an erosion-based system). For example, the drugrelease device can be an electrochemical pump, osmotic pump, anelectroosmotic pump, a vapor pressure pump, or osmotic bursting matrix,e.g., where the drug is incorporated into a polymer and the polymerprovides for release of drug formulation concomitant with degradation ofa drug-impregnated polymeric material (e.g., a biodegradable,drug-impregnated polymeric material). In other embodiments, the drugrelease device is based upon an electrodiffusion system, an electrolyticpump, an effervescent pump, a piezoelectric pump, a hydrolytic system,etc.

Drug release devices based upon a mechanical or electromechanicalinfusion pump can also be suitable for use with the present disclosure.Examples of such devices include those described in, for example, U.S.Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852; and thelike. In general, a subject treatment method can be accomplished usingany of a variety of refillable, non-exchangeable pump systems. Pumps andother convective systems may be utilized due to their generally moreconsistent, controlled release over time. Osmotic pumps are used in someembodiments due to their combined advantages of more consistentcontrolled release and relatively small size (see, e.g., PCTInternational Application Publication No. WO 97/27840 and U.S. Pat. Nos.5,985,305 and 5,728,396). Exemplary osmotically-driven devices suitablefor use in the disclosure include, but are not necessarily limited to,those described in U.S. Pat. Nos. 3,760,984; 3,845,770; 3,916,899;3,923,426; 3,987,790; 3,995,631; 3,916,899; 4,016,880; 4,036,228;4,111,202; 4,111,203; 4,203,440; 4,203,442; 4,210,139; 4,327,725;4,627,850; 4,865,845; 5,057,318; 5,059,423; 5,112,614; 5,137,727;5,234,692; 5,234,693; 5,728,396; and the like. A further exemplarydevice that can be adapted for the present disclosure is the Synchromedinfusion pump (Medtronic).

In some embodiments, the drug delivery device is an implantable device.The drug delivery device can be implanted at any suitable implantationsite using methods and devices well known in the art. As noted herein,an implantation site is a site within the body of a subject at which adrug delivery device is introduced and positioned. Implantation sitesinclude, but are not necessarily limited to a subdermal, subcutaneous,intramuscular, or other suitable site within a subject's body.

Suitable excipient vehicles for a compound disclosed herein are, forexample, water, saline, dextrose, glycerol, ethanol, or the like, andcombinations thereof. In addition, if desired, the vehicle may containminor amounts of auxiliary substances such as wetting or emulsifyingagents or pH buffering agents. Methods of preparing such dosage formsare known, or will be apparent upon consideration of this disclosure, tothose skilled in the art. See, e.g., Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., 17th edition, 1985. Thecomposition or formulation to be administered will, in any event,contain a quantity of the compound adequate to achieve the desired statein the subject being treated.

Compositions of the present disclosure include those that comprise asustained-release or controlled release matrix. In addition, embodimentsof the present disclosure can be used in conjunction with othertreatments that use sustained-release formulations. As used herein, asustained-release matrix is a matrix made of materials, usuallypolymers, which are degradable by enzymatic or acid-based hydrolysis orby dissolution. Once inserted into the body, the matrix is acted upon byenzymes and body fluids. A sustained-release matrix desirably is chosenfrom biocompatible materials such as liposomes, polylactides (polylacticacid), polyglycolide (polymer of glycolic acid), polylactideco-glycolide (copolymers of lactic acid and glycolic acid),polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid,collagen, chondroitin sulfate, carboxcylic acids, fatty acids,phospholipids, polysaccharides, nucleic acids, polyamino acids, aminoacids such as phenylatanine, tyrosine, isoleucine, polynucleotides,polyvinyl propylene, polyvinylpyrrolidone and silicone. Illustrativebiodegradable matrices include a polylactide matrix, a polyglycolidematrix, and a polylactide co-glycolide (co-polymers of lactic acid andglycolic acid) matrix.

In another embodiment, the interfering agent (as well as combinationcompositions) is delivered in a controlled release system. For example,a compound disclosed herein may be administered using intravenousinfusion, an implantable osmotic pump, a transdermal patch, liposomes,or other modes of administration. In one embodiment, a pump may be used(Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al.(1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J. Med. 321:574).In another embodiment, polymeric materials are used. In yet anotherembodiment a controlled release system is placed in proximity of thetherapeutic target, i.e., the liver, thus requiring only a fraction ofthe systemic dose. In yet another embodiment, a controlled releasesystem is placed in proximity of the therapeutic target, thus requiringonly a fraction of the systemic. Other controlled release systems arediscussed in the review by Langer (1990) Science 249:1527-1533.

In another embodiment, the compositions of the present disclosure (aswell as combination compositions separately or together) include thoseformed by impregnation of an inhibiting agent described herein intoabsorptive materials, such as sutures, bandages, and gauze, or coatedonto the surface of solid phase materials, such as surgical staples,zippers and catheters to deliver the compositions. Other deliverysystems of this type will be readily apparent to those skilled in theart in view of the instant disclosure.

The present disclosure provides methods and compositions for theadministration of a one or more of an interfering agent to a host (e.g.,a human) for the treatment of a microbial infection. In variousembodiments, these methods disclosed herein span almost any availablemethod and route suitable for drug delivery, including in vivo and exvivo methods, as well as systemic and localized routes ofadministration.

Screening Assays

The present disclosure provides methods for screening for equivalentagents, such as equivalent monoclonal antibodies to a polyclonalantibody as described herein and various agents that modulate theactivity of the active agents and pharmaceutical compositions disclosedherein or the function of a polypeptide or peptide product encoded bythe polynucleotide disclosed herein. For the purposes of thisdisclosure, an “agent” is intended to include, but not be limited to abiological or chemical compound such as a simple or complex organic orinorganic molecule, a peptide, a protein (e.g., antibody), apolynucleotide anti-sense) or a ribozyme. A vast array of compounds canbe synthesized, for example polymers, such as polypeptides andpolynucleotides, and synthetic organic compounds based on various corestructures, and these are also included in the term “agent.” Inaddition, various natural sources can provide compounds for screening,such as plant or animal extracts, and the like. It should be understood,although not always explicitly stated that the agent is used alone or incombination with another agent, having the same or different biologicalactivity as the agents identified by the inventive screen.

One embodiment is a method for screening agents capable of interactingwith, binding to, or inhibiting the DNA-DNABII (e.g., IHF) interaction.The present disclosure provides in FIG. 6B the three-dimensionalstructure of the microbial DNA and IHF. Accordingly, the disclosurepermits the use of virtual design techniques, also known ascomputer-aided, in silico design or modeling, to design, select, andsynthesize agents capable of interacting with, binding to, or inhibitingthe DNA-DNABII (e.g., IHF) interaction. In turn, the candidate agentsmay be effective in the treatment of biofilms and associated diseases orconditions (medical, industrial or veterinary) as described herein.Thus, the present disclosure also provides agents identified or designedby the in silico methods.

Three-dimensional structure of a IHF-DNA complex is illustrated in FIG.6B and a representative structure, with X, Y and Z coordinates, areprovided in Protein Data Bank Accession Number: 1IHF, with relevantdetails provided in Rice et al. (1996) Cell 87:1295-1306. Thethree-dimensional structure of the IHF protein in the IHF-DNA complexcan be used for the screening method. A suitable agent is one that canbe positioned relative to the IHF protein structure in the IHF-DNAcomplex with interactions at least one, or alternatively two, or three,or four, or five, or six, or seven, or eight, or nine, or at least tenof the amino acid residues that are identified to be involved ininteracting with DNA.

FIG. 6A illustrates the amino acid residues involved in IHF-DNAinteraction, using the E. coli IHF sequence (SEQ ID NO: 42) as anexample. Such amino acid residues, indicated by the lower level ofarrows in FIG. 6A, are further described below, indicated with bold andunderlined letters. Namely, with the E. coli IHF, the amino acidsinvolved in DNA binding are T4, K5, A6, E28, Q43, K45, S47, G48, N51,R55, K57, R60, R63, N64, P65, K66, R76, T80, R82 or Q85.

(SEQ ID NO: 42) MAL TKA EMSE YLFDKLGLSK RDAKELV E LF FEEIRRALEN GE Q V KL SG FG  N FDL R D K NQ R  PG RNPK TGED IPITA R RVV T F R PG QKLKSR VENASPKDE

Thus, one embodiment of the present disclosure provides acomputer-implemented method for identifying an agent that inhibits,competes or titrates the binding of a DNABII polypeptide or protein to amicrobial DNA, that inhibits, prevents or breaks down a microbialbiofilm, that inhibits, prevents or breaks down a biofilm in a subject,or that inhibits, prevents or treats a microbial infection that producesa biofilm in a subject, comprising positioning a three-dimensionalstructure of a candidate agent against a three-dimensional structure ofan integration host factor (IHF) protein, wherein the three-dimensionalstructure of the IHF protein is based on X, Y and Z atomic structurecoordinates determined from a crystalline form of an IHF and DNAcomplex, wherein interaction of the agent with the IHF at two or moreIHF amino acids selected from T4, K5, A6, E28, Q43, K45, S47, G48, N51,R55, K57, R60, R63, N64, P65, K66, R76, T80, R82 or Q85 as representedin SEQ ID NO: 42, or the equivalent of each, identifies that the agentinhibits, competes or titrates the binding of a DNABII polypeptide orprotein to a microbial DNA, inhibits, prevents or breaks down amicrobial biofilm, inhibits, prevents or breaks down a biofilm, orinhibits, prevents or treats a microbial infection that produces abiofilm.

In one aspect, a candidate agent interacts with the IHF protein at leastone, or two, or three of amino acids 63, 64, 65, or 66. In one aspect, acandidate agent interacts with the IHF protein at least one, or two, orthree of R63, N64, P65, K66. In another aspect, a candidate agentinteracts with the IHF protein at least at one of 63 or 66. In anotheraspect, a candidate agent interacts with the IHF protein at least at oneof R63 or K66.

It would be appreciated in the art that the exact locations and aminoacid residues vary depending on the IHF sequence. One of the skill inthe art, however, can readily identify such locations and amino acidresidues based on the sequences. For instance, an IHF sequence can bealigned with the IHF sequence of E. coli (SEQ ID NO: 42), as illustratedin Table 9, to reveal those that correspond to the amino acids in E.coli IHF that interact with DNA. Likewise, the three-dimensionalstructure of such an IHF sequence in an IHF-DNA complex can be used forthe screening.

In addition to the computer-implemented methods as provided herein, thepresent disclosure also provides custom computer system that includes,e.g., processor, memory and/or program, for performing the methods, aswell as a computer readable medium, such as a non-transitory computerreadable medium that stores suitable computer program or code forcarrying out the methods.

Accordingly, another embodiment provides a custom computing apparatuscomprising:

at least one processor;

a memory coupled to the at least one processor;

a storage medium in communication with the memory and the at least oneprocessor, the storage medium containing a set of processor executableinstructions that, when executed by the processor configure the customcomputing apparatus to identify an agent that inhibits, competes ortitrates the binding of a DNABII polypeptide or protein to a microbialDNA, that inhibits, prevents or breaks down a microbial biofilm, thatinhibits, prevents or breaks down a biofilm in a subject, or thatinhibits, prevents or treats a microbial infection that produces abiofilm in a subject, wherein the configuration comprises:positioning a three-dimensional structure of a candidate agent against athree-dimensional structure of an integration host factor (IHF) protein,wherein the three-dimensional structure of the IHF protein is based onX, Y and Z atomic structure coordinates determined from a crystallineform of an IHF and DNA complex, wherein interaction of the agent withthe IHF at two or more IHF amino acids selected from T4, K5, A6, E28,Q43, K45, S47, G48, N51, R55, K57, R60, R63, N64, P65, K66, R76, T80,R82 or Q85 as represented in SEQ ID NO: 42, or the equivalent of each,identifies that the agent inhibits, competes or titrates the binding ofa DNABII polypeptide or protein to a microbial DNA, inhibits, preventsor breaks down a microbial biofilm, inhibits, prevents or breaks down abiofilm, or inhibits, prevents or treats a microbial infection thatproduces a biofilm.

Yet another embodiment provides a non-transitory computer mediumcomprising a set of processor executable instructions that, whenexecuted by a processor, identifying an agent that inhibits, competes ortitrates the binding of a DNABII polypeptide or protein to a microbialDNA, that inhibits, prevents or breaks down a microbial biofilm, thatinhibits, prevents or breaks down a biofilm in a subject, or thatinhibits, prevents or treats a microbial infection that produces abiofilm in a subject, comprising positioning a three-dimensionalstructure of a candidate agent against a three-dimensional structure ofan integration host factor (IHF) protein, wherein the three-dimensionalstructure of the IHF protein is based on X, Y and Z atomic structurecoordinates determined from a crystalline form of an IHF and DNAcomplex, wherein interaction of the agent with the IHF at two or moreIHF amino acids selected from T4, K5, A6, E28, Q43, K45, S47, G48, N51,R55, K57, R60, R63, N64, P65, K66, R76, T80, R82 or Q85 as representedin SEQ ID NO: 42, or the equivalent of each, identifies that the agentinhibits, competes or titrates the binding of a DNABII polypeptide orprotein to a microbial DNA, inhibits, prevents or breaks down amicrobial biofilm, inhibits, prevents or breaks down a biofilm, orinhibits, prevents or treats a microbial infection that produces abiofilm.

Methods of in silico molecule or drug designs are well known in the art,see generally Kapetanovic (2008) Chem Biol. Interact., 171(2):165-76.Briefly, the atomic coordinates of the three-dimensional structure areinput into a computer so that images of the structure and variousparameters are shown on the display. The design typically involvespositioning a three-dimensional structure to the three-dimensionalstructure of the target molecule. The positioning can be controlled bythe user with assistance from a computer's graphic interface, and can befurther guided by a computer algorithm looking for potential goodmatches. Positioning also involves moving either or both of thethree-dimensional structures around at any dimension.

Then, the resultant data are input into a virtual compound and/or agentlibrary. Since a virtual library is contained in a virtual screeningsoftware such as DOCK-4 (Kuntz, UCSF), the above-described data may beinput into such a software. Candidate agents may be searched for, usinga three-dimensional structure database of virtual or non-virtual drugcandidate compounds, such as MDDR (Prous Science, Spain).

A candidate agent is found to be able to bind to DNA and/or DNABIIprotein if a desired interaction between the candidate agent and eitheror both is found. The interaction can be quantitative, e.g., strength ofinteraction and/or number of interaction sites, or qualitative, e.g.,interaction or lack of interaction. The output of the method,accordingly, can be quantitative or qualitative. In one aspect,therefore, the present disclosure also provides a method for identifyingan agent that does not inhibit the interaction or alternatively,strengthens the interaction between the DNA and protein.

The potential inhibitory or binding effect (i.e., interaction orassociation) of an agent such as a small molecule compound may beanalyzed prior to its actual synthesis and testing by the use ofcomputer modeling techniques. If the theoretical structure of the givencompound suggests insufficient interaction and association between itand microbial DNA in the biofilm and/or DNABII protein, synthesis andtesting of the agent can be obviated. However, if computer modelingindicates a strong interaction, the agent can then be synthesized andtested for its ability to bind to or inhibit the interaction usingvarious methods such as in vitro or in vivo experiments. Methods oftesting an agent's ability to inhibit or titrate a biofilm, alone or inconnection with another agent, are disclosed herein. In this manner,synthesis of inoperative agents and compounds can be avoided.

One skilled in the art may use any of several methods to screen chemicalor biological entities or fragments for their ability to associate withDNABII or microbial DNA and more particularly with the specific bindingsites. Selected fragments or chemical entities may then be positioned ina variety of orientations, or docked, within an individual binding siteof DNA or DNABII polypeptide. Docking may be accomplished using softwaresuch as QUANTA, SYBYL, followed by energy minimization and moleculardynamics with standard molecular mechanics force fields, such as CHARMMand AMBER.

Commercial computer programs are also available for in silico design.Examples include, without limitation, GRID (Oxford University, Oxford,UK), MCSS (Molecular Simulations, Burlington, Mass.), AUTODOCK (ScrippsResearch Institute, La Jolla, Calif.), DOCK (University of California,San Francisco, Calif.), GLIDE (Schrodinger Inc.), FlexX (Tripos Inc.)and GOLD (Cambridge Crystallographic Data Centre).

Once an agent or compound has been designed or selected by the abovemethods, the efficiency with which that agent or compound may bind toeach other can be tested and optimized by computational evaluation. Forexample, an effective DNABII fragment or may demonstrate a relativelysmall difference in energy between its bound and free states (i.e., asmall deformation energy of binding).

A compound designed or selected can be further computationally optimizedso that in its bound state it may optionally lack repulsiveelectrostatic interaction with the target protein. Suchnon-complementary (e.g., electrostatic) interactions include repulsivecharge-charge, dipole-dipole, and charge-dipole interactions.Specifically, the sum of all electrostatic interactions between theagent and DNABII and/or microbial DNA in the biofilm when the agent orcompound is bound to either agent, optionally making a neutral orfavorable contribution to the enthalpy of binding.

Computer softwares are also available in the art to evaluate compounddeformation energy and electrostatic interaction. Examples include,without limitation, Gaussian 92 [Gaussian, Inc., Pittsburgh, Pa.]; AMBER[University of California at San Francisco]; QUANTA/CHARMM [MolecularSimulations, Inc., Burlington, Mass.]; and Insight II/Discover [BiosysmTechnologies Inc., San Diego, Calif.].

Once a binding agent has been optimally selected or designed, asdescribed above, substitutions may then be made in some of its atoms orside groups in order to improve or modify its binding properties.Generally, initial substitutions are conservative, i.e., the replacementgroup will have approximately the same size, shape, hydrophobicity andcharge as the original group. It should, of course, be understood thatcomponents known in the art to alter conformation should be avoided.Such substituted chemical compounds may then be analyzed for efficiencyof fit to the DNABII protein and/or microbial DNA in the biofilm by thesame computer methods described in detail, above.

Certain embodiments relate to a method for screening small moleculescapable of interacting with the protein or polynucleotide disclosedherein. For the purpose of this disclosure, “small molecules” aremolecules having low molecular weights (MW) that are, in one embodiment,capable of binding to a protein of interest thereby altering thefunction of the protein. In some embodiments, the MW of a small moleculeis no more than 1,000. Methods for screening small molecules capable ofaltering protein function are known in the art. For example, aminiaturized arrayed assay for detecting small molecule-proteininteractions in cells is discussed by You et al. (1997) Chem. Biol.4:961-968.

To practice the screening method in vitro, suitable cell culture ortissue infected with the microbial to be treated are first provided. Thecells are cultured under conditions (temperature, growth or culturemedium and gas (CO₂)) and for an appropriate amount of time to attainexponential proliferation without density dependent constraints. It alsois desirable to maintain an additional separate cell culture that is notinfected as a control.

As is apparent to one of skill in the art, suitable cells can becultured in micro-titer plates and several agents can be assayed at thesame time by noting genotypic changes, phenotypic changes or a reductionin microbial titer.

When the agent is a composition other than a DNA or RNA, such as a smallmolecule as described above, the agent can be directly added to the cellculture or added to culture medium for addition. As is apparent to thoseskilled in the art, an “effective” a mount must be added which can beempirically determined,

When the agent is an antibody or antigen binding fragment, the agent canbe contacted or incubated with the target antigen and polyclonalantibody as described herein under conditions to perform a competitiveELISA. Such methods are known to the skilled artisan.

The assays also can be performed in a subject. When the subject is ananimal such as a rat, chinchilla, mouse or simian, the method provides aconvenient animal model system that can be used prior to clinicaltesting of an agent in a human patient. In this system, a candidateagent is a potential drug if symptoms of the disease or microbialinfection is reduced or eliminated, each as compared to untreated,animal having the same infection. It also can be useful to have aseparate negative control group of cells or animals that are healthy andnot treated, which provides a basis for comparison.

The agents and compositions can be used in the manufacture ofmedicaments and for the treatment of humans and other animals byadministration in accordance with conventional procedures, such as anactive ingredient in pharmaceutical compositions.

Combination Therapy

The compositions and related methods of the present disclosure may beused in combination with the administration of other therapies. Theseinclude, but are not limited to, the administration of DNase enzymes,antibiotics, antimicrobials, or other antibodies.

In some embodiments, the methods and compositions include adeoxyribonuclease (DNase) enzyme that acts synergistically with theanti-DNABII antibody. A DNase is any enzyme that catalyzes the cleavageof phosphodiester linkages in the DNA backbone. Three non-limitingexamples of DNase enzymes that are known to target not only cruciformstructures, but also a variety of secondary structure of DNA includeDNAse I, T4 EndoVII and T7 Endo I. In certain embodiments, the effectiveamount of anti-DNABII antibody needed to destabilize the biofilm isreduced when combined with a DNase. When administered in vitro, theDNase can be added directly to the assay or in a suitable buffer knownto stabilize the enzyme. The effective Unit dose of DNase and the assayconditions may vary, and can be optimized according to procedures knownin the art.

In other embodiments, the methods and compositions can be combined withantibiotics and/or antimicrobials. Antimicrobials are substances thatkill or inhibit the growth of microorganisms such as bacteria, fungi, orprotozoans. Although biofilms are generally resistant to the actions ofantibiotics, compositions and methods described herein can be used tosensitize the infection involving a biofilm to traditional therapeuticmethods for treating infections. In other embodiments, the use ofantibiotics or antimicrobials in combination with methods andcompositions described herein allow for the reduction of the effectiveamount of the antimicrobial and/or biofilm reducing agent. Somenon-limiting examples of antimicrobials and antibiotics useful incombination with methods of the current disclosure include amoxicillin,amoxicillin-clavulanate, cefdinir, azithromycin, andsulfamethoxazole-trimethoprim. The therapeutically effective dose of theantimicrobial and/or antibiotic in combination with the biofilm reducingagent can be readily determined by traditional methods. In someembodiments the dose of the antimicrobial agent in combination with thebiofilm reducing agent is the average effective dose which has beenshown to be effective in other bacterial infections, for example,bacterial infections wherein the etiology of the infection does notinclude a biofilm. In other embodiments, the dose is 0.1, 0.15, 0.2,0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.8,0.85, 0.9, 0.95, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5,3.0 or 5 times the average effective dose. The antibiotic orantimicrobial can be added prior to, concurrent with, or subsequent tothe addition of the anti-DNABII antibody.

In other embodiments, the methods and compositions can be combined withantibodies that treat the bacterial infection. One example of anantibody useful in combination with the methods and compositionsdescribed herein is an antibody directed against an unrelated outermembrane protein (i.e., OMP P5). Treatment with this antibody alone doesnot debulk a biofilm in vitro. Combined therapy with this antibody and abiofilm reducing agent results in a greater effect than that which couldbe achieved by either reagent used alone at the same concentration.Other antibodies that may produce a synergistic effect when combinedwith a biofilm reducing agent or methods to reduce a biofilm includeanti-rsPilA anti-OMP26, anti-OMP P2, and anti-whole OMP preparations.

The compositions and methods described herein can be used to sensitizethe bacterial infection involving a biofilm to common therapeuticmodalities effective in treating bacterial infections without a biofilmbut are otherwise ineffective in treating bacterial infections involvinga biofilm. In other embodiments, the compositions and methods describedherein can be used in combination with therapeutic modalities that areeffective in treating bacterial infections involving a biofilm, but thecombination of such additional therapy and biofilm reducing agent ormethod produces a synergistic effect such that the effective dose ofeither the biofilm reducing agent or the additional therapeutic agentcan be reduced. In other instances the combination of such additionaltherapy and biofilm reducing agent or method produces a synergisticeffect such that the treatment is enhanced. An enhancement of treatmentcan be evidenced by a shorter amount of time required to treat theinfection.

The additional therapeutic treatment can be added prior to, concurrentwith, or subsequent to methods or compositions used to reduce thebiofilm, and can be contained within the same formation or as a separateformulation.

Kits

Kits containing the agents and instructions necessary to perform the invitro and in vivo methods as described herein also are claimed.Accordingly, the disclosure provides kits for performing these methodswhich may include an interfering disclosed herein as well asinstructions for carrying out the methods disclosed herein such ascollecting tissue and/or performing the screen, and/or analyzing theresults, and/or administration of an effective amount of an interferingagent as defined herein. These can be used alone or in combination withother suitable antimicrobial agents.

For example, a kit can comprise, or alternatively consist essentiallyof, or yet further consist of any one or more agent identified above,e.g., an agent of the group of an isolated or recombinant DNABIIpolypeptide, an integration host factor (IHF) polypeptide or a fragmentor an equivalent of each thereof; an isolated or recombinant proteinpolypeptide identified in Table 8, Table 9, Table 9 a DNA bindingpeptide identified in FIGS. 6A-6B, or a fragment or an equivalent ofeach thereof; an isolated or recombinant polypeptide of SEQ ID NO: 1through 33, or a fragment or an equivalent of each thereof; an isolatedor recombinant polypeptide of SEQ ID NO: 342 through 353, or a fragmentor an equivalent of each thereof; an isolated or recombinant C-terminalpolypeptide of SEQ ID NO: 5 through 11, 28, 29, 340 or those identifiedin Table 8, Table 9 or a fragment or an equivalent of each thereof; apolypeptide that competes with an integration host factor on binding toa microbial DNA; a four-way junction polynucleotide resembling aHolliday junction, a 3 way junction polynucleotide resembling areplication fork, a polynucleotide that has inherent flexibility or bentpolynucleotide; an isolated or recombinant polynucleotide encoding anyone of the above noted polypeptides; an antibody that specificallyrecognizes or binds any one of the above noted polypeptides, or anequivalent or fragment thereof; or a small molecule that competes withthe binding of a DNABII protein or polypeptide to a microbial DNA, andinstructions for use. The kit can further comprising one or more of anadjuvant, an antigenic peptide or an antimicrobial. Examples of carriersinclude a liquid carrier, a pharmaceutically acceptable carrier, a solidphase carrier, a pharmaceutically acceptable carrier, a pharmaceuticallyacceptable polymer, a liposome, a micelle, an implant, a stent, a paste,a gel, a dental implant, or a medical implant.

TABLE 8 Gram (+) - only HU, Gram (−) - all have HU some also IHFBacteria strain Abbreviation Protein name(s) S. sobrinus 6715 Ss 1310(HU) (not fully sequenced) S. pyogenes MGAS10270 Spyog Spy1239 (HU) S.gordonii Challis NCTC7868 Sg SGO_0701 (HlpA) S. agalactiae (Group BStrep) 2603V/R GBS SAG_0505 (Hup) S. mutans UA159 Sm Smu_589 (HU) S.pneumoniae R6 Spneu spr1020 (HU) S. gallolyticus UCN34 (S. bovis) SgallYP_003430069 (HlpA) S. aureus MW2 So MW1362 (HU) S. epidermidis RP62A SeSERP1041 (Hup) E. coli K12-MG1655 Ec b1712 (HimA) b0912 (HimD) (HupA)(HupB) H. influenza KW20 Rd Hi HI1221 (HimA) HI1313 (HimD) HI0430 (HupA)Salmonella enteric serovar typhi CT18 Salm Sty1771 (HimA) Sty0982 (HimD)Aggregatibacter Aa YP_003255965 (IHFalpha) actinomycetemcomitans D11S-1YP_003256209 (IhfB) YP_003255304 (HU) P. gingivalis W83 Pg PG 0121(Hup-1) PG_1258 (Hup-2) N. gonorrhoeae FA1090 (Oklahoma) Ng NGO603(IHFβ) NGO030 (IHFα) N. meningitides MC58 Nm NMB_0729 (HimA) NMB_1302(HimA) P. aeruginosa Pa PA3161 (HimD) PA1804 (HupB) PA2758 (HimA) H.pylori 26695 Hp Hp0835 (Hup) B. burgdorferi B31 Bb BB_0232 (Hbb)Moraxella catarrhalis RH4 Mc YP_003626307 (HimA) YP_003627027 (HimD)YP_003626775 (HupB) V. cholera El Tor N16961 Vc VC_0273 (HupA) VC_1914(HipB) VC_1919 (HupB) VC_1222 (HimA) Burkholderia cenocepacia HI2424 BcBcen2424_1048 (IHFB) Bcen2424_1481 (IHFA) Burkholderia pseudomallei 668Bp BURPS668_2881 (IHFB) BURPS668_1718 (IHFA) Mycobacterium tuberculosisCDC1551 Mtb MT_3064 (HU) Mycobacterium smegmatis MC2 Ms MSMEG_2389 (Hup)Treponema denticola ATCC 35405 Td TDE_1709 (HU) Treponema palladumNichols Tp TP_0251 (DNA binding protein II) Prevotella melaninogenicaATCC 25845 Pm PREME0022_2103 (HupB) PREME0022_0268 (HupA) PREME0022_0341(Hup) PREME0022_0340 (HimA) Prevotella intermedia 17 Pi PIN_A0704 (Hup)PIN_A1504 (Hup-2) PIN_0345 (HimA) PIN_0343 (Hypothetical protein)Bordetella pertusis Tohama 1 Bpert BP2572 (IhfA) BP3530 (HupB) BP0951(IhfB) Enterococcus faecalis V583 Ef Ef1550 (hup)

TABLE 9 (SEQ ID NOS 160-336, respectively, in order of appearance)Bacteria strain, protein name β3 sequence α3 sequence C-terminal 20 aaS. pyogenes MGAS10270, HU AFKAGKALKDAVK IAASKVPAFKAGKALKDAVKS. gallolyticus UCN34 (S. AFKAGKALKDAVK IAASKVPAFKAGKALKDAVKbovis), HlpA S. sobrinus 6715 HU AFKAGKALKDAVK IAASKVPAFKAGKALKDAVKS. agalactiae (Group B AFKAGKALKDAVK IAASKVPAFKAGKALKDAVKStrep)2603V/R Hup S. pneumoniae R6 HU AFKAGKALKDAVK IAASKVPAFKAGKALKDAVKS. gordonii Challis NCTC7868, AFKAGKALKDAVK IAASKVPAFKAGKALKDAVK HlpAS. mutans UA159, HU AFKAGKALKDAVK IKASKVPAFKAGKALKDAVKEnterococcus faecalis V583, AFKPGK ALKDAVK IAASKVPAFKPGKALKDAVK HupS. aureus MW2, HU AFKAGKALKDAVK IPASKVPAFKAGKALKDAVKS. epidermidis RP62A Hup AFKAGKALKDAVK IPASKVPAFKAGKALKDAVKH. influenza KW20 Rd HupA AFVSGK ALKDAIK IAASKVPAFVSGKALKDAIKAggregatibacter AFVSGK ALKDAVK IAASKVPAFVSGKALKDAVKactinomycetemcomitans D11S-1 HU V. cholera El Tor N16961, AFVAGKALKDAIKIAAANVPAFVAGKALKDAIK HupA E. coli K12-MG1655 hupA AFVSGK ALKDAVKIAAANVPAFVSGKALKDAVK P. aeruginosa HupB GFKAGKALKDAVNIAAAKIPGFKAGKALKDAVN E. coli K12-MG1655 hupB SFRAGK ALKDAVNIAAAKVPSFRAGKALKDAVN V. cholera El Tor N16961 SFKAGK ALKDACNIAEAKVPSFKAGKALKDACN HupB Bordetella pertusis Tohama 1 KFRPGK ALKDAVNIKKAKVPKFRPGKALKDAVN HupB Prevotella melaninogenica KFKAGAELADAVNKAAKKVAKFKAGAELADAVNK ATCC 25845 HupB Prevotella intermedia 17 HupKFKPGA ELADAVNA AAKKVAKFKPGAELADAVNA Moraxella catarrhalis RH4SFKAGK VLKESVN IAASKVPSFKAGKVLKESVN HupB P. gingivalis W83 Hup-1RFKPGS TLELK ISIPARKVVRFKPGSTLELK H. pylori 2669 Hup KFKPGK TLKQKVEEGKKRVPKFKPGKTLKQKVEEGK Prevotella melaninogenica SFKPAK TFIEDMKKPAHDFPSFKPAKTFIEDMKK ATCC 25845 HupA Prevotella intermedia 17 Hup-2SFKPAK TFIEDMKK PAHDFPSFKPAKTFIEDMKK P. gingivalis W83 Hup-2AFKPSK IFMSQMKQD KRNIPAFKPSKIFMSQMKQD Mycobacterium tuberculosisAFRPGA QFKAVVSGAQRLPAEGPAVKRG AKRPATKAPAKKATARRGRK CDC1551 HUMycobacterium smegmatis MC2 AFRPGA QFKAVISGAQKLPADGPAVKRGTKAPAKKAAAKKAPAKKGRR Hup Prevotella melaninogenica NFKPAA TIKGHVRKGGQDNGNFKPAATIKGHVRKGGQDNG ATCC 25845 HimA Prevotella intermedia 17 HimANFRATA SVKEKLKKGGAE VLNFRATASVKEKLKKGGAE E. coli K12-MG1655 HimATFRPGQ KLKSRVENASPKDE TFRPGQKLKSRVENASPKDE Salmonella enteric serovarTFRPGQ KLKSRVENASPKEE TFRPGQKLKSRVENASPKEE typhi CT18 HimAV. cholera El Tor N1696 HimA TFRPGQ KLKARVENIKVEK VTFRPGQKLKARVENIKVEKP. aeruginosa HimA TFRPGQ KLKARVEAYAGTKS TFRPGQKLKARVEAYAGTKSBurkholderia cenocepacia TFHASQ KLKALVENGAE RVVTFHASQKLKALVENGAEHI2424 IHFA Burkholderia pseudomallei 668 TFHASQ KLKALVENGAEPDLARHASQKLKALVENGAEPDLAR IHFA Bordetella pertusis Tohama 1TFHASQ KLKSVVEQPNSPPDPASAE QKLKSVVEQPNSPPDPASAE IhfAN. gonorrhoeae FA1090 TFHASQ KLKGMVEHYYDKQR TFHASQKLKGMVEHYYDKQR(Oklahoma) IHFα N. meningitides MC58 HimA TFHASQ KLKSMVEHYYDKQRTFHASQKLKSMVEHYYDKQR H. influenza KW20 Rd HimA TFKPGQ KLRARVEKTKRRVVTFKPGQKLRARVEKTK Aggregatibacter VFKPGQ KLRNRVEKVKPKAVVFKPGQKLRNRVEKVKPKA actinomycetemcomitans D11S-1 HimAMoraxella catarrhalis RH4 TFKAGQKLRGWIDSQNEG VVTFKAGQKLRGWIDSQNEG HimATreponema palladium Nichols VFRPSK RLKSAVRGYRSGEVGADPSKRLKSAVRGYRSGEVGAD DNA_binding_protein_II Prevotella melaninogenicaSFTPDT VMKELVNKPFSQFETVVINDGV MQAGDTMKVPKVELRPEYRK ATCC 25845 HupPrevotella intermedia 17 SFTPDA TMKELVNKPFAQFETVVLNDGVSAGDTMKVPKVELRPQYRTK hypothetical E. coli K12-MG1655 HimDHFKPGK ELRDRANIYG KYVPHFKPGKELRDRANIYG Salmonella enteric serovarHFKPGK ELRDRANIYG KYVPHFKPGKELRDRANIYG typhi CT18 bHimDV. cholera El Tor N1696 HipB HFKPGK ELRERVNL EGKYVPHFKPGKELRERVNLP. aeruginosa HimD HFKPGK ELRDRVNEPE KFVPHFKPGKELRDRVNEPEH. influenza KW20 Rd HimD YFKAGKELKARVDVQA KSVPYFKAGKELKARVDVQAAggregatibacter YFKAGKELRERVDVYAA CVPYFKAGKELRERVDVYAAactinomycetemcomitans D11S-1 IHFB N. gonorrhoeae FA1090HFKPGK ELRERVDLALKENAN FKPGKELRERVDLALKENAN (Oklahoma) IHFβN. meningitides MC58 HimD HFKPGK ELRERVDLALKENAN FKPGKELRERVDLALKENANBurkholderia cenocepaciaHFKPGK ELRERVDGRAGEPLKADDPDDDRERVDGRAGEPLKADDPDDDR HI2424 IHFBBurkholderia pseudomallei 668 HFKPGK ELRERVDGRAGEPLKNDEPEDAQERVDGRAGEPLKNDEPEDAQ IHFB Bordetella pertusis Tohama 1HFKAGKELREWVDL VGNDQGDDSSNGSS DSSNGSSDPLQSVMDMHAMH IhfBMoraxella catarrhalis RH4 YFKPGK ALRESVNLVND ATPYFKPGKALRESVNLVND HimDB. burgdorferi B31 Hbb YFRPGK DLKERVVVGIKG HVAYFRPGKDLKERVVVGIKGTreponema denticola ATCC RFKPGK ELKEALHKIDTQELIES PGKELKEALHKIDTQELIES35405 HU

The following examples are intended to illustrate, and not limit theembodiments disclosed herein.

EXPERIMENTAL Experiment No. 1

Some of the IHF antibodies, proteins and polypeptides were a generousgift from Nash. The methods to produce them are well known to theskilled artisan, e.g., as described in Granston and Nash (1993) J. Mol.Biol. 234:45-59; Nash et al. (1987) Journal of Bacteriology169(9):4124-4127; and Rice et al. (1996) Cell 87:1295-1306. Briefly, tooverproduce IHF-α, the himA gene was inserted downstream from the P_(L)promoter in the bacterial plasmid pAD284. Transformants of strain K5607,a lambda lysogen of strain C600himA42 that had received the desiredplasmid, were identified by screening ampicillin-resistant transformantsfor the ability to grow bacteriophage Mu (13). DNA was prepared fromhimA⁺ transformants according to standard DNA isolation techniques, andthe orientation of the himA gene was determined by restriction enzymecleavage. Plasmid pP_(L)himA-1, which has the himA gene in the properorientation for expression by the P^(L) promoter, was transformed intostrain N5271, which contains a cryptic lambda prophage expressing thecI857 thermoinducible repressor, to yield strain K5770.

To overproduce IHF-β, plasmid pKT23-hip323, which contains a fusion ofthe IHF-β coding sequence to the bacteriophage lambda P_(L) promoter wasused. pKT23-hip323 was introduced into N5271 to give strain E443. Tofacilitate the selection of pKT23-hip323 in the presence of anotherplasmid, changed its selectable marker was changed from ampicillinresistance (bla⁺) to chloramphenicol resistance (cat⁻). A cat-containingfragment was isolated from plasmid pBR325 as described by Flamm andWeisberg and was inserted into the unique ScaI site in bla. The ligatedDNA was introduced into strain E403, which carries a hip mutation andwhich synthesizes temperature-sensitive X repressor, andchloramphenicol-resistant transformants were selected at lowtemperature. One such transformant (E735) was hip⁺ and ampicillinsensitive; it therefore appears to carry a bla cat⁺ derivative ofpKT23-hip323 (pE735).

To generate a strain that overproduces both subunits of IHF, E735 wastransformed with plasmid pP_(L)himA-1, selecting transformant (E738)that had become ampicillin resistant and had retained chloramphenicolresistance. The generation of a second strain that overproduces bothsubunits of depended on the construction of plasmid pP_(L)hip himA-5,which was made by ligating blunted (SstII restriction enzyme site)containing the pheT and himA genes into the pKT23-hip323 plasmid. Thisis described in further detail in (Nash et al. (1987) J. Bacteriology169(9):4124-4127. himA⁺ transformants of strain K5607 were identified byscreening for HimA⁺, and the plasmid DNA was analyzed by restrictiondigestion. In all cases where the plasmid structure was obvious, twocopies of himA had been ligated as a tandem direct repeat into thevector. It is not known if the presence of two copies of the himA geneon this plasmid is demanded by the selection, but it should be recalledthat a single copy of the himA gene in plasmid pP_(L)himA-1 issufficient to complement a himA mutant. Plasmid pP_(L)hiphimA-5 was usedto transform strain N5271 to yield strain K5746.

Cells were grown in shaking water bath at 31° C. in TBY medium (10 g oftryptone, 5 g of yeast extract, and 5 g of sodium chloride per liter).At mid-log phase (optical density at 650 nm, ca. 0.6), the cells wereshifted to a 42° C. water bath and shaking was continued. Typically, 300ml of culture was centrifuged and suspended in 0.6 to 0.9 ml of TEG (20mM Tris hydrochloride (pH 7.4), 1 mM sodium EDTA, 10% glycerol)containing 20 mM NaCl. The cells were disrupted with six 20-s bursts ofsonication, with 40 s between each burst. A portion of the sonic extractwas centrifuged in a Sorvall SS34 rotor for 20 min at 15,000 rpm.Samples of the sonic extract were analyzed by sodium dodecyl sulfate(SDS) gel electrophoresis according to standard molecular biologytechniques.

Purification of was done according to the following: A 3.6-liter batchof cells was induced for 3 h. All subsequent steps were carried out at 0to 4° C. The cell pellet from 3.3-liters was suspended in 10 ml of TEGcontaining 20 mM NaCl to give a total volume of 29 ml; this suspensionwas disrupted in two batches, each receiving six bursts of 3 min ofsonication separated by 90-s intervals. The sonic extract wascentrifuged for 20 min at 15,000 rpm, yielding 16.9 ml of clarifiedextract. A 10% (vol/vol) solution (1.1 ml) of polymin P (BDH ChemicalsLtd.) was added slowly to the clarified extract; after being stirred for20 mm, the mixture was centrifuged for 30 mm at 10,000 rpm. Theresulting pellet was suspended in 10 ml of TEG containing 500 mM NaCl;after being stirred for 15 min, the mixture was centrifuged for 20 minat 12,000 rpm. The supernatant (10.3 ml) was adjusted to 50% saturationby the addition of 3.2 g of ammonium sulfate, stirred for 20 min, andcentrifuged for 15 min at 15,000 rpm. The resulting supernatant wasadjusted to 70% saturation by the addition of 1.64 g of ammoniumsulfate, stirred for 20 min, and centrifuged for 15 min at 15,000 rpm.The resulting pellet was suspended in 1 ml of TG (50 mM Trishydrochloride (pH 7.4) containing 10% glycerol) and dialyzed against twochanges of TG. The dialyzed material (2.0 ml) was loaded onto a 1-mlcolumn (0.5 by 5.8 cm) of phosphocellulose (P11; Whatman, Inc.) that hadbeen equilibrated with TG. The column was washed with 3 ml of TG anddeveloped with 20 ml of a linear gradient (0 to 1.2 M) of KCl in TG.Fractions of 0.5 ml were collected, stored at −20° C., and assayed forIHF activity.

Polyclonal anti-IHF was prepared as follows. Rabbits were injected with250 μg of purified IHF with Freund's complete adjuvant. Boosterimmunizations of 250 μg of IHF with Freund's incomplete adjuvant weregiven 1, 7, and 12 weeks later. As determined by immunoblotting of IHF,sera collected 13 weeks after the initial injection had a high titer ofIHF-reactive material. The animals were maintained for several yearsand, when necessary, given further booster immunization in order tomaintain a high titer of anti-IHF in their sera. The antibody was notpurified further. Crude sera was stored at −70° C.

Antibodies specific to each subunit were generated by using syntheticpolypeptides corresponding to the most C-terminal 20 amino acid residuesof each subunit (SEQ ID NO: 34 and 35), to immunize rabbits according toDitto et al. (1994) J. Bacteriology 176(12):3738-3748.

Experiment 2

This experiment describes an in vitro model for reversal of anestablished biofilm in 8-well chamber slide. The materials used in thisexperiment were: Chocolate Agar; sBHI (BHI with 2 mg home/mL and 2 mgb-NAD/mL); 8-well Chamber slides (Nunc* Lab-Tek* Fisher catalog#12-565-18); Sterile 0.9% saline; LIVE/DEAD BacLight Bacterial ViabilityKit (Fisher catalog #NC9439023) and Formalin.

On day 1, NTHI was struck for isolation on chocolate agar. It was thenincubated for 20 hrs at 37° C. and 5% CO₂. The next day, bacteria weresuspended in 5 mL equilibrated (37° C., 5% CO₂) and optical density wasadjusted to OD₄₉₀=0.65 in sBHI. Bacteria were diluted 1:6 inequilibrated sBHI (1 mL bacterial suspension+5 mL sBHI). Bacteria wasthen incubated for 3 hours at 37° C. in 5% CO₂, static (OD₄₉₀ should beapprox 0.65). Next, the bacteria were again diluted 1:2500 inequilibrated sBHI and 200 mL of the bacterial suspension was added toeach well of the chamber slide. For dilution, 10 μL bacteria was addedto 990 μL sBHI in an eppendorf tube and 8 μL dilution was added to 192μL sBHI in each chamber and incubated at 37° C., 5% CO₂, static.

On the third day after 16 hours of incubation medium was aspirated fromchamber by aspirating medium from the corner of the well so as not todisturb biofilm. Then 200 mL of equilibrated sBHI was added to eachchamber and incubated for 37° C., 5% CO₂, static for 8 hours. After 8hours, the medium was aspirated and 200 mL equilibrated sBHI was addedto each untreated chamber; and 200 mL of interfering agent such asRabbit anti-rsPilA; diluted 1:50 in sBHI and 200 mL Naive rabbit serum(or other appropriate serum control) diluted 1:50 in sBHI was added.They were then incubate at 37° C. and 5% CO₂, static.

On day 4, after approximately 16 hours of incubation, aspirate sBHI wasaspirated and the biofilm was washed twice with 200 mL sterile saline.The saline was aspirated and 200 mL Live/Dead stain was added. Next, 3mL component A plus 3 mL component B in 1 mL sterile 10 mM phosphatebuffered saline was added. It was then incubated for 15 minutes at roomtemperature, static, protected from light. Stain was aspirated andbiofilm was washed twice with 200 mL sterile saline. Saline wasaspirated and 200 mL formalin was added to fix biofilm. It was thenincubated 15 minutes at room temperature, static, protected from light.Formalin was aspirated and biofilm was washed twice with 200 mL sterilesaline. Gasket was removed and coverslip were placed on slide; coverslipwere sealed with nail polish and dried prior to viewing by confocalmicroscopy.

Using this method and anti-IHF antibody described in Experiment No. 1,Applicants reduced a biofilm produced by Haemophilus influenzae which isprevalent in sinusitis, bronchitis, otitis media and exacerbations ofchronic obstructive pulmonary disease (COPD). Untreated biofilm mass wasmeasured to be 4.24 μm³/μm² with a mean thickness of 11.68 μm. Aftertreatment, the biofilm mass was reduced to 0.53 μm³/μm² with a meanthickness of 1.31 μm. Thus, this shows an 88.8% reduction in meanthickness and an 87.5% reduction in biomass.

Polyclonal antisera directed against the E. coli IHF raised against anIHF promoter purified from E. coli K12 was prepared in rabbits accordingto standard techniques using purified Integration Host Factor (IHF).Experiment 1 describes the expression and purification of IHF from E.coli. This antibody is the anti-IHF antibody used to generate the datain the experiments disclosed below.

Using this method and anti-IHF antibody, Applicants reduced a biofilmproduced by Streptococcus mutans which is prevalent in initiation andprogression of dental caries. Untreated biofilm mass was measured to be1.17 μm³/μm² with a mean thickness of 5.43 μm. After treatment, thebiofilm mass was reduced to 0.1 μm³/μm² with a mean thickness of 0.47μm. Thus, this shows a 91.3% reduction in mean thickness and an 91.6% %reduction in biomass. In vitro biofilm assays were repeated 3 times, onseparate days. The percent reduction in the max height, averagethickness, and biomass is depicted in Table 1 below.

TABLE 1 Assay 1 Assay 2 Assay 3 S. mutans Max height (μm) 56 24 41 Avethickness (μm) 65 65 67 biomass (μm³/μm²) 37 58 66

Using this method and anti-IHF antibody, Applicants reduced a biofilmproduced by Staphylococcus aureus which is prevalent in localized anddiffuse skin infections, chronic rhinosinusitis and nosocomialinfections. Untreated biofilm mass was measured to be 0.3 μm³/μm² with amean thickness of 2.2 μm and a biofilm height of 17.5 μm. Aftertreatment, the biofilm mass was reduced to 0.3 μm³/μm² with a meanthickness of 1.1 μm and a biofilm height of 8 μm. Thus, this shows a48.8% reduction in mean thickness and a 2.7% reduction in biomass (FIG.7 ).

Using this method and anti-IHF antibody, Applicants reduced a biofilmproduced by Moraxella catarrhalis which is prevalent in exacerbation ofCOPD and otitis media. Untreated biofilm mass was measured to be 0.72μm³/μm² with a mean thickness of 1.48 μm. After treatment, the biofilmmass was reduced to 0.13 μm³/μm² with a mean thickness of 0.65 μm. Thus,this shows a 55.8% reduction in mean thickness and an 82.1% reduction inbiomass. In vitro biofilm assays were repeated 3 times, on separatedays. The percent reduction in the max height, average thickness, andbiomass is depicted in Table 2 below.

TABLE 2 Assay 1 Assay 2 Assay 3 M. catarrhalis Max height (μm) 61 33 36Ave thickness (μm) 92 34 84 biomass (μm³/μm²) 92 35 92

Using this method and anti-IHF antibody, Applicants reduced a biofilmproduced by Streptococcus pneumoniae which is prevalent in sinusitis,pneumonia and otitis media. Untreated biofilm mass was measured to be0.64 μm³/μm² with a mean thickness of 3.99 μm. After treatment, thebiofilm mass was reduced to 0.14 μm³/μm² with a mean thickness of 0.82μm. Thus, this shows a 79.5% reduction in mean thickness and a 78.6%reduction in biomass. In vitro biofilm assays were repeated 3 times, onseparate days. The percent reduction in the max height, averagethickness, and biomass is depicted in Table 3 below.

TABLE 3 Assay 1 Assay 2 Assay 3 S. pneumoniae Max height (μm) 49 51 44Ave thickness (μm) 25 64 79 biomass (μm³/μm²) 51 61 79

Using this method and anti-IHF antibody, Applicants reduced a biofilmproduced by Pseudomonas aeruginosa which is prevalent in cysticfibrosis, pneumonia, skin and soft tissue infections and on medicaldevices. Untreated biofilm mass was measured to be 7.0 μm³/μm² with amean thickness of 25.70 μm and a biofilm height of 40 μm. Aftertreatment, the biofilm mass was reduced to 3.4 μm³/μm² with a meanthickness of 10.3 μm and a biofilm height of 27.5 μm. Thus, this shows a60.1% reduction in mean thickness and a 50.8% reduction in biomass (FIG.7 ).

Using this method and anti-IHF antibody, Applicants reduced a biofilmproduced by Neisseria gonorrhoeae which is in gonorrhea. Untreatedbiofilm mass was measured to be 9.5 μm³/μm² with a mean thickness of22.02 μm and a biofilm height of 40 μm. After treatment, the biofilmmass was reduced to 0.8 μm³/μm² with a mean thickness of 3.4 μm and abiofilm height of 27.5 μm. Thus, this shows an 84.5% reduction in meanthickness and a 92.1% reduction in biomass (FIG. 7 ).

Using this method and anti-IHF antibody, Applicants reduced a biofilmproduced by Uropathogenic E. coli which is prevalent in urinary tractinfections. Untreated biofilm mass was measured to be 1.75 μm³/μm² witha mean thickness of 31.73 μm. After treatment, the biofilm mass wasreduced to 0.75 μm³/μm² with a mean thickness of 1.62 μm. Thus, thisshows a 94.9% reduction in mean thickness and a 56.9% reduction inbiomass. In vitro biofilm assays were repeated 3 times, on separatedays. The percent reduction in the max height, average thickness, andbiomass is depicted in Table 4 below.

TABLE 4 Assay 1 Assay 2 Assay 3 UPEC Max height (μm) 69 65 76 Avethickness (μm) 97 33 95 biomass (μm³/μm²) 98 96 57

Using this method and anti-IHF antibody, Applicants reduced a biofilmproduced by Staphylococcus epidermidis which is prevalent in infectionsof the skin. In vitro biofilm assays were repeated 3 times, on separatedays. The percent reduction in the max height, average thickness, andbiomass is depicted in Table 5 below.

TABLE 5 Assay 1 Assay 2 Assay 3 S. epidermidis Max height (μm) 42 38 56Ave thickness (μm) 62 71 92 biomass (μm³/μm²) 62 76 88

Experiment No. 3

Middle ear infection (or otitis media, OM) is a highly prevalent diseaseworldwide, afflicting 50-330 million children globally each year. Thesocioeconomic burden of OM is also great, with cost estimates between$5-6 billion in the United States alone annually. All three of thepredominant bacterial pathogens of OM are known to form biofilms both invitro and in vivo and recently, clinicians have come to appreciate thatthe chronicity and recurrence of OM is due, at least in part, to theformation of bacterial biofilms within the middle ear cavity. In onechinchilla model of OM, juvenile chinchillas are first given a viral‘cold’, followed a week later by their being challenged intranasallywith an inoculum viable bacteria. Similar to the human condition wherein“my child has a cold and a week later gets an ear infection” chinchillaswill also develop a bacterial OM approximately one week after achallenge, and while experiencing the viral upper respiratory tractinfection. Once bacteria gain access to the middle ear (either viaascension of the Eustachian tube or following direct challenge to themiddle ear space), they will form a robust biofilm. Applicants thuscontemplated and indeed have already used chinchilla models as reportedherein to demonstrate the protective efficacy of IHF immunization whichresults in rapid resolution of existing biofilms. This model is alsouseful for therapeutic approaches via either passive delivery ofanti-DNABII antibody or via delivery of a small molecule or other agentknown to bind to IHF or other DNABII family members.

Because the chinchilla model is used for development and pre-clinicaltesting of human vaccines, it is important to establish meaningfulimmunological parallels with the human host, particularly the child.Applicants have shown that effusions recovered from children with AOMdue to NTHI, and middle ear fluids from chinchillas with experimentalNTHI-induced OM, recognized immunodominant regions of OMP P5 in asimilar hierarchical manner (see for e.g., Novotny, L. A. et al. (2000)Infect 68(4):2119-28; Novotny, L. A. et al. (2007) 9^(th) InternationalSymposium on Recent Advances in Otitis Media; St. Pete Beach, Fla.;Novotny, L. A. et al. (2002) Vaccine 20(29-30):3590-97). Applicants havealso shown that chinchillas with experimental OM, children with naturalOM, and adults with exacerbations of COPD, all recognized peptidesrepresenting PilA in a highly analogous manner (see for e.g., Adams, L.D. et al. (2007) 107th General Meeting, American Society forMicrobiology; 2007; Toronto, ON; Adams, L. D. et al. (2007) 9thInternational Symposium on Recent Advances in Otitis Media; St. PeteBeach, Fla.). Thus, chinchillas with experimental OM and children withnatural disease respond similarly immunologically to at least twounrelated NTHI protein adhesins. This parallel was put to the ultimatetest recently, when the chinchilla AV-NTHI superinfection model was usedto conduct pre-clinical efficacy testing of a novel 11-valent ProteinD-pneumococcal polysaccharide conjugate vaccine. Data obtained in thechinchilla predicted an efficacy of 34% whereas, when tested inchildren, the efficacy obtained against H. influenzae-induced OM was35.6% (see for e.g., Novotny, L. A. et al. (2006) Vaccine 24(22):4804-11and Prymula, R. et al. (2006) Lancet. 367(9512):740-8), thus lendingstrong support to the relevancy of this model for the development andtesting of OM vaccine candidates.

Applicants have shown a dramatic reduction in the pre-formed biofilmremaining within the middle ears of chinchillas after receipt of 2weekly TC immunizations with an IHF promoter purified from E. coli K12(the full length combination of both IHF subunits) delivered with amucosal/systemic adjuvant.

48 adults were ordered from Rauscher's Chinchilla Ranch (LaRue, Ohio)and were acclimated to the vivarium for 7-10 days prior to the beginningof the study. Prior to TB [transbullar or direct challenge into themiddle ear cavity] challenge, baseline otoscopy and tympanometry wereperformed as well as a limited volume prebleed to collect serum.Chinchillas were anesthetized and 300 μl of NTHI (strain #86-028NP)suspension containing approximately 1000 cfu were introduced into themiddle ear space via transbullar challenge. Animals were allowed torecover, then monitored daily for adverse reactions as per IACUCaccepted animal use protocols. Routine otoscopy and tympanometry wereperformed every 2-3 days throughout the study.

Four days after challenge (day +4), chinchillas were anesthetized and aCT scan was performed to visualize biofilms present within the middleear and to obtain pre-immunization images. The surface of the pinnaewere cleaned and hydrated by wiping with a sterile gauze pad moistenedwith sterile pyrogen free saline. After ^(˜)2 minutes (to allow thepinnae to dry), 50 μl of either the dmLT, IHF (purified E. coli IHFaccording to Experiment 1)+dmLT, or IHF+rsPiLA+dmLT solution will beadded to the pinna and gently massaged in. Three chinchillas per cohortwere sacrificed and tissues/samples collected (on days +4, and +11) tobegin to determine mechanism of action.

Eleven days after challenge (day +11), the secondary immunizationoccurred as described above wherein the pinnae are cleaned/hydrated andimmunogens were topically administered. Three chinchillas per cohortwere sacrificed and tissues/samples collected to begin to determinemechanism of action.

Eighteen days after challenge (day +18), chinchillas were anesthetizedand a CT scan were performed to identify any biofilms present, as wellas to compare to the pre-immunization CT scans. The animals were thenbled to collect serum and euthanized. The bullae are dissected and anyfluids present were aseptically collected, the bullae were then openedto visualize the inferior bulla and any biofilm present. Images werecollected and the bulla were washed with 1 ml of sterile saline andre-imaged. The mucosa, along with any biofilm present, were collectedfrom the right bulla and placed in a preweighed tube. These tissues werehomogenized, serially diluted, and plated to determine cfu NTHI/mg wetweight tissue. The left bullae were filled with OCT compound and snapfrozen for histological analysis.

Images of the left and right middle ear cavities, with residentbiofilms, were scrambled and two images per animal were compiled into asingle file for ranking by blinded evaluators. The relative amount ofbiomass remaining within the middle ear of each animal was ranked on a 0to 4+ scale by blinded reviewers using the scale shown in Table 6 below.Titer ELISAs, cytokine arrays, and Biacore were run on the serum as wellas on the collected middle ear effusions. The OCT embedded middle earswere sectioned and stained either for basic morphology and architecture(H&E) or for the presence of IHF using immunohistochemistry and/orimmunofluorescence.

TABLE 6 Score Criteria 0 No evidence of biomass.  1+ Biomass fills ≤25%of middle ear space. Junction of the bony septa to inferior bulla isvisible.  2+ Biomass tills >25% to ≤50% of middle ear space. Unable tovisualize where the bony septa meet the inferior bulla.  3+ Biomassfills >50% to ≤75% of middle ear space. Biomass covers >50% of thelength of bony septa.  4+ Biomass tills >75% to ≤100% of middle earspace. Bony septa ot visible; obscured by biomass.

Immunofluorescence imaging of frozen sections of biofilms formed in vivowas preformed according to the following. After dissection, the middleear of each chinchilla was filled with OCT embedding compound (FisherScientific, Pittsburgh, Pa.) and flash frozen over liquid nitrogen. Thebone which forms the inferior bulla was removed to leave the middle earmucosa and existing biofilm intact. The resulting block was thenbisected to reveal a cross section of the biofilm and re-embedded inOCT. Ten micron serial sections were cut using a Microm rotary cryotome,adhered to glass slides (Mercedes Medical, Sarasota, Fla.) and stored at−80° C. Sections were later stained to determine the relativeincorporation of IHF within biofilms that had formed in vivo. Briefly,slides were air-dried, fixed in cold acetone, then equilibrated inbuffer (0.05M Tris-HCl, 0.15M NaCl and 0.05% Tween 20, pH 7.4). Sectionswere blocked with image-iT FX signal enhancer (Molecular Probes, Eugene,Oreg.) and with Background Sniper (BioCare Medical, Concord, Calif.) permanufacturer's instructions. Sections were then incubated with a 1:200dilution of polyclonal rabbit anti-IHF overnight at 4° C., in ahumidified chamber. Slides were further rinsed and incubated with goatanti-rabbit IgG conjugated to AlexaFluor 594 (Invitrogen) for 30 minutesat room temp. As a counterstain, sections were incubated with DAPI andcover-slipped using ProLong Gold antifade reagent (Molecular Probes,Eugene, Oreg.). Naive rabbit serum served as the negative control.Sections were viewed with a Zeiss LSM 510 Meta confocal system attachedto a Zeiss Axiovert 200 inverted microscope (Carl Zeiss Inc., Thornwood,N.Y.).

Significant differences in mean CFU/mg tissue and mean CFU NTHI/mlsupernatant were determined by paired t-test. A p-value ≤0.05 wasconsidered significant. Significance in relative biomass among cohortswas assessed by unpaired t-test. A p-value ≤0.05 was consideredsignificant.

As shown in FIG. 9A, the mean score for remaining biomass within themiddle ears of chinchillas immunized with adjuvant only was 2.8 whichindicated significant remaining disease and a lack of resolution ofpre-formed biomass in most animals by day 18 after bacterial challengeof the middle ear. In contrast, the mean score for an E. coli IHF+adjuvant immunized animal was 1.5. Representative images of a residualbiomass that received a mean score of +2.8 and one that received a meanscore of +1.5 are shown in FIG. 9B. Also, as shown in FIGS. 10A-10B, thedisease resolution was additionally measured by both histologicalevidence of altered biomass architecture within the middle ear (see FIG.10A) as well as a statistically significant reduction of bacterial loadpresent within remaining biomass as measured by homogenization of thebiomass and culture on chocolate agar (see FIG. 10B). Furthermore, allanimals immunized with isolated E. coli IHF mounted a strong local andsystemic immune response and no animal presented with obvious secondarysequelae as the result of immunization as noted upon necropsy (data notshown).

The notable observed efficacy when anti-IHF raised against an IHFpromoter purified from E. coli K12 (the full length combination of bothIHF subunits) used in vitro to debulk biofilms and also of purified E.coli IHF, when used as an immunogen in vivo to induce the formation ofpolyclonal antibodies that could resolve an ongoing biofilm disease,created a conundrum as to why mammalian hosts do not naturally mount aneffective immune response to DNABII proteins that are associated witheDNA within the bacterial biofilms of recurrent and/or chronic diseasestates. In review of the solved structure of IHF when it is bound to DNA(Rice et al. (1996) Cell 87(7):1295-1306), it is clear that asignificant portion of the protein structure is occluded by bound DNA,which suggested the potential for occlusion of protective epitopes ordomains of IHF and/or HU when bound to eDNA within a bacterial biofilm.It was hypothesized that use of native IHF, to which no DNA was bound,as the immunogen might provide a mechanism to overcome such occlusionand thereby foster production of protective antibodies.

To determine if eDNA could indeed prevent the development of protectiveantibodies directed against a DNABII family member upon immunization,whereas use of native protein was effective, a second larger cohortstudy was performed wherein the chinchilla study as detailed previouslywas essentially repeated.

For the second animal immunization study, twenty adult chinchillas (bodymass between 500-700 g), shown to have no evidence of middle ear diseaseby tympanometry and video otoscopy, were enrolled and divided into fourcohorts of 5 animals each. All animals were again challengedtransbullarly with approximately 1000 CFU NTHI strain 86-028NP perbulla. Four days later, after a biofilm formed in the middle earcavities of these animals, they were immunized by a transcutaneous route(TCI) as described above. Formulations consisted of either: 10 μg IHFadmixed with 10 μg dmLT, 10 μg IHF that had been pre-bound to DNA+dmLT,DNA+dmLT, or 10 μg dmLT alone.

To determine if TCI with IHF pre-bound to DNA resulted in a similarimmune response to that induced when the same nucleoprotein complex wasdelivered subcutaneously (SQ), and compared to that induced by IHFalone, two adult chinchillas were immunized to generate antisera. Eachanimal received a priming dose followed by two identical boostsdelivered at 30-day intervals. Immunogens were admixed with the adjuvantmonophosphoryl lipid A (MPL) (10 μg/dose) (Sigma-Aldrich, St. Louis,Mo.) due to its demonstrated strong adjuvant properties in thechinchilla host (See for example Bakaletz et al. (1999) Infect Immun.67(6):2746-2762 and Kennedy et al. (2000) Infect. Immun.68(5):2756-2765. One chinchilla was immunized with 10 μg of IHF+MPL andthe other received 10 μg IMF bound to DNA+MPL. All doses were deliveredSQ in a total volume of 200 Fifteen days after receiving the finalboost, animals were bled to procure serum and sera were assayed viaWestern blot and ELISA to determine both reciprocal titer andspecificity of antibody reactivity to IHF.

Middle ears were again subsequently scored from 0 to 4+ for diseaseseverity upon completion of the study. To better assure that the IHF andDNA remained in complex for immunization, synthetic oligonucleotidesidentical to those used in the published co-crystallization study (seefor e.g., Rice et al. (1996) Cell 87(7):1295-1306) of a high affinityIHF binding site from the bacteriophage lambda recombination site attPat a molar ratio of 2:1 [DNA (10 μM) to IHF (5 μM)] was used. Thisamount was at least 3 orders of magnitude over the K_(d) of IHF bound tothis DNA target. As shown in FIG. 11 , IHF indeed bound dsDNA asdemonstrated by its reduced mobility in an electrophoretic mobilityshift assay.

As shown in FIG. 12 , animals immunized with isolated E. coli IHF showeda dramatic reduction in disease state with a mean residual middle earbiomass score of 0.9 as compared to the controls that had been immunizedwith adjuvant alone (mean biomass score=2.2) or to those that had beenimmunized with DNA that had been admixed with adjuvant (mean biomassscore=2.8). Interestingly, those animals that had been immunized withthe IHF-DNA complex also demonstrated middle ears with significantremaining bacterial biomass, yielding a mean biomass score of 2.5 whichwas not statistically significantly different from cohorts that receivedeither the adjuvant alone or DNA that had been admixed with adjuvant.This outcome strongly suggested that if sufficient eDNA fragments werepresent, as one could hypothesize would be the case during naturaldisease, this situation could result in occlusion of critical IHFepitopes necessary for the generation of neutralizing antibodies.

To be assured that the observed DNA occlusion result was not specific tothe use of a transcutaneous immunization route, these immunizationsusing a subcutaneous (SQ) immunization route to ensure delivery of theantigens to antigen presenting cells within the chinchilla host wererepeated. As shown in FIG. 13 , whereas SQ immunization with isolated E.coli IHF yielded the generation of a strong immune response to isolatedIHF, immunization with E. coli IHF that had been pre-bound to an excessof DNA failed to induce detectable antibodies that recognized IHF whenassayed by Western blot. When assayed by ELISA, reciprocal titer versusisolated IHF was 1000 for the animal immunized with IHF that had beenpre-bound to oligonucleotides, whereas that for the animal immunizedwith native IHF was 8000 (both animal's pre-immune reciprocal titersagainst IHF were 100). Collectively, these results are consistent withour hypothesis that the binding of IHF to eDNA, as would occur during anatural disease state, has the potential to block epitopes or domains ofIHF necessary for generation of a protective acquired immune response.Further, immunization with native IHF (to which no DNA is bound)appeared to allow for the effective direction of the immune responsetoward the generation of protective or neutralizing antibodies, asdemonstrated in both pre-clinical chinchilla studies described here.

Experiment No. 4

A number of oral bacteria (e.g., Aggregatibacter actinomycetemcomitans,Porphyromonas gingivalis) have been implicated in the pathogenesis ofinflammatory diseases such as periodontitis and peri-implantitis, whichdestroy alveolar bone and gingiva. Investigations of the pathogenesis ofthese bacteria are hampered by lack of effective animal models. One ofthe challenges of investigating the pathogenicity of specific bacteriais the difficulty of establishing a biofilm when exogenous bacteria areintroduced into the oral cavity of animals. Though animal models ofperiodontitis have been developed, cultivable bacteria are rarelyrecovered from the oral cavity of inoculated animals. Developing aneffective animal model which can assess the pathogenicity of specificbacteria wilt greatly aid in elucidating their pathogenic mechanisms.

The surface of machined titanium dental implants (1.2×4.5 mm) wasmodified by grit blasting with A103 (100 μm) and HCl etching (pH 7.8 for20 min at 80° C.). Machined and nano-textured implants were incubated inTSB medium inoculated with D7S clinical strain of Aggregatibacteractinomycetemcomitans (Aa) for 1 to 3 days at 37° C. The bacterialbiofilm on the implants were analyzed by SEM, as well as by confocallaser scanning microscopy following staining with LIVE/DEAD® BacLight™.Implants with and without established Aa biofilm were transmucosallyplaced into the alveolar bone of female rats between premolar andincisor region of the maxillae. To detect the presence of Aa biofilm onthe implants placed in vivo, bacterial samples were collected fromsaliva and the oral surfaces of implants after 2 days. Aa was detectedby culture, as well as by PCR analysis. Micro-CT and histologicalanalysis of peri-implant bone and mucosal tissues was performed sixweeks after implantation.

After one day of cultivation, agglomerates of coccoid-shaped Aa cellswere found scattered throughout the implant. After two days, the numberand size of the agglomerates decreased and more cells of varying lengthswere observed ranging between bacteria with coccoid morphology. Afterthree days of incubation, the agglomerates had almost disappeared, whilelarge areas of the implant surface were covered with bacteria withrod-shaped morphology, forming a densely packed biofilm. LIVE/DEAD®staining of such three days Aa biofilm on the implants showed greensignal for 75-80% of all biofilm bacteria, indicating living cells withuncompromised membrane integrity. Microbiological and PCR detection ofAa biofilm on implants placed in vivo were positive for samples from dieimplant surfaces and negative for the saliva samples as well as controlimplants. Clinical examination demonstrated significant peri-implantmucosal inflammation around implants with Aa biofilm, compared withcontrol untreated implants. Micro-CT and histological analysis ofperi-implant bone and mucosal tissues is pending. Nano-textured implantsurfaces favor the establishment of Aa biofilm and increase risk ofperi-implantitis.

Experiment No. 5

This experiment provides a mouse model for pre-clinical testing ofinterfering agents to treat lyme disease. See Dresser et al. Pathogens5(12)e1000680, Epub 2009 Dec. 4. Lyme disease is the most commontick-borne disease in the United States. Reported cases have more thandoubled between 1992 and 2006, with approximately 29,000 new casesconfirmed in 2008. Estimates are that the actual number of cases of Lymedisease may exceed that reported by a factor of 6-12 in endemic areas.By definition, these endemic areas are expanding as populations continueto move from cities to suburban and rural areas and whitetail deer(which carry the tick species Ixodes) increasingly roam these areas.Lyme disease is caused by the microorganism Borrelia burgdorferi, aspirochete. B. burgdorferi is transmitted via the bite of the Ixodestick and subsequently disseminates, via the bloodstream, to othertissues and organs.

In this animal model, C3H/HeN mice are injected with spirochetes viadorsal subcutaneous and intraperitoneal injection, or via intravenousinjection. Blood and biopsy specimens are recovered at approximately 7days post infection for evaluation of microbial burden and assessment ofpathology in tissues and organs. The methods and compositions disclosedherein are contemplated to develop both therapeutic as well aspreventative strategies for reduction and/or elimination of theresulting B. burgdorferi biofilms which form subsequent to challenge andare believed to contribute to both the pathogenesis and chronic natureof the disease.

Experiment No. 6

This experiment provides a porcine model for pre-clinical testing ofinterfering agents to treat cystic fibrosis. See Stoltz et al. (2010)Science Translational Medicine 2(29):29-31. Cystic fibrosis is anautosomal recessive disease due to mutations in a gene that encodes theCF transmembrane conductance regulator (called CFTR) anion channel. Inthis model, pigs which have been specifically bred to carry a defect inthe genes called “CFTR” and called CF pigs spontaneously develophallmark features of CF lung disease that includes infection of thelower airway by multiple bacterial species. The pigs can be immunizedwith the interfering agents to either 1) immunize these CF pigs with apolypeptide or other immunogenic agent thereby inducing the formation ofantibodies which will eradicate bacterial biofilms in the lungs(similarly to how antibodies to IHF eradicated biofilms resident withinthe middle ears of chinchillas following active immunization as shown inExperiment No. 1, to deliver anti-IHF (or other interfering agent) tothe lungs of these animals by nebulization to assess the amelioration ofthe signs of disease and associated pathologies.

Experiment No. 7

Applicants also provide a pre-clinical model for tuberculosis (TB). SeeOrdway et al. (2010) Anti. Agents and Chemotherapy 54:1820. Themicroorganism Mycobacterium tuberculosis is responsible for a growingglobal epidemic. Current figures suggest that there are approximately 8million new cases of TB and about 2.7 million deaths due to TB annually.In addition to the role of this microbe as a co-infection of individualswith HIV (of the ^(˜)45 million infected with HIV, estimates are that^(˜)⅓ are also co-infected with M. tuberculosis), its particularlytroublesome that isolates have become highly resistant to multiple drugsand no new drug for TB has been introduced in over a quarter of acentury. In this animal model, SPF guinea pigs are maintained in abarrier colony and infected via aerosolized spray to deliver ^(˜)20 cfuof M. tuberculosis strain Erdman K01 bacilli into their lungs. Animalsare sacrificed with determination of bacterial load and recovery oftissues for histopathological assessment on days 25, 50, 75, 100, 125and 150 days post-challenge. Unlike mice which do not develop classicsigns of TB, guinea pigs challenged in this manner developwell-organized granulomas with central necrosis, a hallmark of humandisease. Further, like humans, guinea pigs develop severepyogranulomatous and necrotizing lymphadenitis of the draining lymphnodes as part of the primary lesion complex. Use of this model willprovide a pre-clinical screen to confirm and identify therapeutic aswell as preventative strategies for reduction and/or elimination of theresulting M. tuberculosis biofilms which have been observed to form inthe lungs of these animals subsequent to challenge and are believed tocontribute to both the pathogenesis and chronicity of the disease.

Experiment No. 8

Multiple animal models of catheter/indwelling device biofilm infectionsare known. See Otto (2009) Nature Reviews Microbiology 7:555. Whiletypically considered normal skin flora, the microbe Staphylococcusepidermidis has become what many regard as a key opportunistic pathogen,ranking first among causative agents of nosocomial infections.Primarily, this bacterium is responsible for the majority of infectionsthat develop on indwelling medical devices which are contaminated bythis common skin colonizer during device insertion. While not typicallylife-threatening, the difficulty associated with treatment of thesebiofilm infections, combined with their frequency, makes them a seriouspublic health burden. Current costs associated with treatment ofvascular catheter associated bloodstream infections alone that are dueto S. epidermidis amount to $2 billion annually in the United States. Inaddition to S. epidermidis, E. faecalis and S. aureus are alsocontaminations found on indwelling medical devices. There are severalanimal models of catheter-associated S. epidermidis infections includingrabbits, mice, guinea pigs and rats all of which are used to study themolecular mechanisms of pathogenesis and which lend themselves tostudies of prevention and/or therapeutics. Rat jugular vein cathetershave been used to evaluate therapies that interfere with E. faecalis, S.aureus and S. epidermidis biofilm formation. Biofilm reduction is oftenmeasured three ways—(i) sonicate catheter and calculate CFUs, (ii) cutslices of catheter or simply lay on a plate and score, or (iii) thebiofilm can be stained with crystal violet or another dye, eluted, andOD measured as a proxy for CFUs.

Experiment No. 9

Methods described herein may be used to elicit immune responses inhumans and animals. Immunogenic compositions may be administered to ahuman and animal subjects in the presence of adjuvants such as but notlimited to aluminum salts and liposomes. Those skilled in the art willunderstand that any number of pharmaceutically acceptable adjuvants canalso be used. Immunogenic compositions may be administered to a human oranimal subjects intramuscularly, subdermally, intranasally, or throughany other suitable route. Immunogenic compositions may be prepared in amanner consistent with the selected mode of administration. Immunogeniccompositions may take the form of polypeptides, nucleic acids, or acombination thereof, and may comprise full-length or partial antigens.Additionally or alternatively, immunogenic compositions may take theform of APCs pulsed with a particular antigen, or APCs transfected withone or more polynucleotides encoding a particular antigen.Administration may comprise a single dose of an immunogenic composition,or an initial administration, followed by one or more booster doses.Booster doses may be provided a day, two days, three days, a week, twoweeks, three weeks, one, two, three, six or twelve months, or at anyother time point after an initial dose. A booster dose may beadministered after an evaluation of the subject's antibody titer.

Experiment No. 10

Methods described herein may be used to confer passive immunity on anon-immune subject. Passive immunity against a given antigen may beconferred through the transfer of antibodies or antigen bindingfragments that specifically recognize or bind to a particular antigen.Antibody donors and recipients may be human or non-human subjects.Additionally or alternatively, the antibody composition may comprise anisolated or recombinant polynucleotide encoding an antibody or antigenbinding fragment that specifically recognizes or binds to a particularantigen.

Passive immunity may be conferred in cases where the administration ofimmunogenic compositions poses a risk for the recipient subject, therecipient subject is immuno-compromised, or the recipient subjectrequires immediate immunity. Immunogenic compositions may be prepared ina manner consistent with the selected mode of administration.Compositions may comprise whole antibodies, antigen binding fragments,polyclonal antibodies, monoclonal antibodies, antibodies generated invivo, antibodies generated in vitro, purified or partially purifiedantibodies, or whole serum. Administration may comprise a single dose ofan antibody composition, or an initial administration followed by one ormore booster doses. Booster doses may be provided a day, two days, threedays, a week, two weeks, three weeks, one, two, three, six or twelvemonths, or at any other time point after an initial dose. A booster dosemay be administered after an evaluation of the subject's antibody titer.

Experiment 11

Members of the DNABII family are highly pleiotropic for multiplenucleoprotein systems including gene transcription, and moreover, arethus often essential. In contrast, both HU and IHF mutants have beengenerated in laboratory strains of E. coli and studied extensively. Todetermine what role IHF and HU have in eDNA formation, biofilms formedby E. coli strain MG1655 or its HU and IHF deficient derivatives wereincubated with anti-IHF (prepared as described in Granston and Nash(1993) J. Mol. Biol. 234:45-59.) As shown in FIGS. 8A-8F, whereas theparental isolate, strain MG1655 produced a robust biofilm in vitro (FIG.8A), both HU and IHF mutant strains were less robust (FIGS. 8C and 8E,respectively). An HU deficient mutant yielded a biofilm that wasapproximately ½ the height of the wild type biofilm, whereas IHFdeficient strains produced a biofilm that was approximately ⅔rds theheight of the parental isolate. This result suggested that both proteinswere involved in either the production and/or integrity of the E. colibiofilm EPS. After treatment with anti-IHF, the biofilm formed by thewild type isolate showed notable debulking and a mean percent reductionof 58.1% in height, 73.1% percent reduction in biomass and 87% reductionin mean thickness (FIG. 8B), whereas that formed by the HU mutant showedno reduction in height and only a 30.9% reduction on biomass and 37.2%reduction on mean thickness (FIG. 8D). In contrast, no effect on thebiofilm formed by the IHF mutant was observed in terms of loss of height(−3.4% reduction), biomass (−20.5% reduction) or mean thickness (−19.8%reduction) (FIG. 8F). Collectively, these data indicated that for thisstrain of E. coli, IHF was likely the only structural element that couldbe targeted by use of the anti-IHF antibody. Since to date only thehighly structured interwoven eDNA in biofilms that have been formed invivo have been seen, confirmation of the expression of remaining DNABIIfamily member by each respective mutant and any change in the resultantDNA structure awaits an in vivo immunofluorescent analysis.

Experiment 12

Whereas it was demonstrated in this application in vitro and in vivothat both anti-IHF and the use of IHF as an immunogen show utility indebulking biofilms and/or resolving biofilm disease respectively, it wasunknown if dispersing of NTHI biofilms with anti-IHF might also allowsynergism when used in conjunction with other therapeutic modalities. Tothis end the ability to induce augmented structural destabilization ofpre-formed NTHI biofilms was assessed when a sub-optimal concentrationof anti-IHF was used along with one of each of three unique reagents.These three reagents include 1) a DNA degrading enzyme (DNaseI) known tobe able to degrade an NTHI biofilm in vitro (but used here at asuboptimal concentration), 2) antisera to an outer membrane protein ofNTHI that did not destabilize a pre-formed NTHI biofilm when used alone(anti-OMP P5) (data not shown), and 3) an antibiotic typically used as afirst line choice in children with chronic and/or recurrent OM(amoxicillin) but which has limited efficacy against bacteria residentwithin a biofilm community.

FIG. 14A shows that treatment of an NTHI biofilm with a concentration ofDNase shown to be sub-optimal has a marginal effect (see FIG. 14A, PanelII). Likewise, a 1:200 dilution of anti-IHF had little effect on thepre-formed NTHI biofilm (see FIG. 14A, Panel III). In contrast however,when these two reagents were used in concert, the biofilm was notablydiminished (see FIG. 14A, Panel IV). Upon repeat of this experimentthree times, we found that the most marked synergistic effect ofdebulking of the biofilm was measured as a diminution in height. Table 7below depicts these results.

TABLE 7 DNase + DNase Anti-IHF anti-IHF Max height (μm) 3.9 37.3 51.0Assay 1 Max height (μm) −11.5 16.4 37.7 Assay 2 Max height (μm) −1.619.4 38.7 Assay 3

One simple explanation for this outcome was that as the DNABII proteinwas being titrated away from the periphery of the biofilm, the eDNAbecame more accessible to the action of the DNase.

FIG. 14B shows the results of treatment with anti-OMP P5 on pre-formedNTHI biofilms. Although this antisera is strongly reactive with isolatedNTHI OMP P5 (data not shown) and further, active immunization withisolated OMP P5 is effective in mediating significant protection againstexperimental OM in chinchilla models (see for e.g., Bakaletz et al.(1997) Vaccine 15(9):955-961; Bakaletz et al. (1999) Infect Immun67(6):2746-2762; Kennedy et al. (2000) Infect Immun 68(5):2756-2765;Kyd, J. M. et al. (2003) Infect Immun 71(8):4691-4699; Novotny, L. A. etal. (2000) Infect Immun 68(4):2119-2128; Novotny, L. A. et al. (2002)Vaccine 20(29-30):3590-3597; Novotny, L. A. et al. (2003) J Immunol171(4):1978-1983) this antiserum does not induce a change in thestructural integrity of an NTHI biofilm that has been formed in vitrowhen used at a dilution of 1:50 (see FIG. 14B, Panel II). Likewise amarginal effect when these biofilms were incubated with a sub-optimaldilution of anti-IHF (used at a 1:100 dilution here) was observed (seeFIG. 14B, Panel I). When combined, however, the use of anti-P5 plusanti-IHF resulted in a reduction in the height of the biofilm thatexceeded the sum of the two antisera when used singly (see FIG. 14B,Panel III), thus indicating a synergistic outcome. Hence, it wasconcluded that use of anti-IHF to destabilize the NTHI EPS matrixresulted in the exposure of the targeted bacterial cell surface protein(e.g., OMP P5) that would otherwise be obscured by eDNA as well asperhaps other components of the EPS, thus allowing immune mediatedbacterial clearance by as yet unknown mechanisms.

Lastly, FIG. 14C shows the results of treating pre-formed NTHI biofilmswith amoxicillin. When used at a concentration of 64 μg/ml, amoxicillinhad no measurable effect on the architecture of pre-formed NTHI biofilms(see FIG. 14C, Panel II) despite evidence of limited bacterial celldeath. Treatment with IHF antisera at a 1:50 dilution substantiallyreduced the height of the biofilm as shown previously (see FIG. 14C,Panel III). Interestingly however, when the two reagents were usedsimultaneously, not only was there a dramatic reduction in the height ofthe biofilm (see FIG. 14C, Panel IV), but use of a vital dye indicatedthat the majority of the bacteria remaining in the biofilm were now deadas noted by the predominant fluorescence in the red channel within theimaged biofilm. This result showed that debulking of the biofilm withanti-IHF likely exposed the bacteria sufficiently so as to createconditions more akin to susceptibility to amoxicillin concentrationsknown to be effective when assayed against planktonic NTHI. This outcomemay have been mediated by either increased physical exposure of bacteriawithin the remaining biofilm matrix to the action of amoxicillin and/orvia increased release of bacteria into the planktonic phase as we showedearlier can occur during biofilm debulking by exposure to anti-IHFantibodies (see FIGS. 4A-4B).

Lastly, FIG. 14C shows the results of treating pre-formed NTHI biofilmswith amoxicillin. When used at a concentration of 64 μg/ml, amoxicillinhad no measurable effect on the architecture of pre-formed NTHI biofilms(see FIG. 14C, Panel II) despite evidence of limited bacterial celldeath. Treatment with IHF antisera at a 1:50 dilution substantiallyreduced the height of the biofilm as shown previously (see FIG. 14C,Panel III). Interestingly however, when the two reagents were usedsimultaneously, not only was there a dramatic reduction in the height ofthe biofilm (see FIG. 14C, Panel IV), but use of a vital dye indicatedthat the majority of the bacteria remaining in the biofilm were now deadas noted by the predominant fluorescence in die red channel within theimaged biofilm. This result showed that debulking of the biofilm withanti-IHF likely exposed the bacteria sufficiently so as to createconditions more akin to susceptibility to amoxicillin concentrationsknown to be effective when assayed against planktonic NTHI. This outcomemay have been mediated by either increased physical exposure of bacteriawithin the remaining biofilm matrix to the action of amoxicillin and/orvia increased release of bacteria into the planktonic phase as we showedearlier can occur during biofilm debulking by exposure to anti-IHFantibodies (see FIGS. 4A-4B).

Experiment 13

Applicants sought to identify the immunodominant and immunoprotectivedomains of IHF 20-mer peptides with a 5-mer overlap were prepared fromthe deduced amino acid sequences of the genes that encode for IHF and HUin nontypeable Haemophilus influenzae (NTHI) strain 86-028NP (disclosedin FIG. 18 ). These peptides mimic the deduced amino acid sequence fromN- to C-terminus but do not accommodate for either discontinuous orconformational epitopes within the properly folded native protein. Grossepitope mapping was performed using antiserum derived from chinchillasthat had been parenterally immunized with native IHF isolated from E.coli+ (the full length combination of both IHF subunits) and an adjuvantin order to determine if immunization with this protein would induce theformation of antibodies that could resolve pre-existing biofilms thathad been formed in their middle ears by NTHI strain 86-028NP. Theimmunization was efficacious, thus inducing the formation of antibodiesthat eradicating experimental otitis media significantly earlier thancontrols which had received adjuvant only. Using BIACORE biosensor, the20-mer peptides and antiserum from these animals, it was determined thatthe greatest immune recognition was to peptides that representedpeptides A5 and B4 (from the alpha and beta subunit respectively). Thiswas unexpected, as individuals suffering from biofilm diseases do notresolve biofilms thus suggesting that the observed result was atherapeutically induced environment Applicants considered how DNABIIproteins would be available for responding to immunologically by theimmune system in an individual with active disease wherein the DNABIIproteins would most likely be complexed with eDNA within the biofilmmatrix. Without being bound by theory, it was hypothesized that sinceIHF or HU would be complexed with eDNA in the biofilm during disease,this might mask the immunoprotective domains of the protein, thus makingthem unavailable to the immune system for recognition and generation ofantibodies.

To test this hypothesis, additional cohorts of chinchillas wereimmunized with either native full length IHF from E. coli (obtained fromNash) or with native full length IHF from E. coli (obtained from Nash)that had been complexed to an excess of double stranded DNA in order tobest mimic how it would be presented in nature. Again, using BIACOREbiosensor, the 20-mer peptides and antiserum from these two cohorts ofanimals, it was again determined that for those animals immunized withnative IHF, the greatest recognition was to peptides A5 and B4, however,when using serum derived from animals immunized with IHF that had beenpre-complexed to an excess of DNA (to best mimic how the immune system“sees” this protein during disease, the greatest recognition was topeptides A3 and B2. Typically, for development of a novel vaccine ortherapeutic, one epitope maps a protein based on how it is presentedduring disease, then one identifies the most immunodominant epitopes ofthat protein and uses these to attempt to design a vaccine candidate orimmune target for treatment.

However, the epitope mapping data indicates that targeting the mostimmunodominant epitopes of IHF as available for responding toimmunologically during disease for either vaccine or therapeuticdevelopment would be a flawed approach. Instead, it was necessary toimmunize with epitopes that are not available for responding toimmunologically during disease in order to redirect the immune system tothe appropriate and immunoprotective targets. This would not have beendetermined had Applicants not immunized chinchillas with both nativeprotein and protein that had been pre-complexed to an excess of DNA andperformed both efficacy studies and comparative epitope mapping efforts.

Once these unexpected protective domains were identified, Applicantsperformed more fine mapping studies to better understand themechanism(s) that underlied the results. To do so, the sequences of the20-mer peptides were overlaid upon a theoretical 3D model of IHF or HUfrom NTHI (the deduced amino acid sequences of these NTHI proteins weresubstituted for those from E. coli for which a 3D crystal structure hasalready been obtained). It was found that the peptides A3 (amino acids31 to 50 of IHF; SEQ ID NO: 350) and B2 (amino acids 16 to 35 of IHF;SEQ ID NO: 343) mapped to the “tails” of IHF whereas the peptides A5(amino acids 61 to 80 of IHF; SEQ ID NO: 352) and B4 (46 to 65 of IHF;SEQ ID NO: 345) mapped to the ‘tips’ of the IHF protein. These datashowed that during disease, the immune system likely only ‘sees’ thetails of these proteins as the tips are bound to eDNA and thus maskedfrom the immune system. This observation would be counterintuitive towhat is typically done to determine the most immunodominant epitope of aprotein as presented by a bacterial pathogen during disease.

The generated 20-mer peptide were simply made in a sequence from N- toC-terminus, e.g., amino acids 1 to 20, 16 to 35, etc., and did notnecessarily mimic the most optimal peptide sequence in terms of bestreproducing either the structure of a discontinuous epitope or the3-dimensional structure of these proteins (the latter of which isessential for their ability to bind to and bend DNA). Therefore, whenneeded, the amino acid sequence of a given 20-mer peptide was modifiedto better fit the new information obtained from the prior study on howthese proteins interact with DNA, combined with our new understandingthat the immunodominant epitopes of DNABII proteins bound to DNA werenot the ones that would likely mediate a protective effect.

Experiment No. 14

Antibodies were raised in chinchillas against the IHFA and IHFB ofHaemophilus influenzae according to the methods described in ExperimentNo. 1 specific to the polypeptide epitopes disclosed in SEQ ID NO: 342to 353.

Using the in vitro model for reversal of an established biofilmdescribed in Experiment 2 and these polyclonal antibodies, Applicantsreduced a biofilm produced by NTHI. The disruption of the biofilm usingthe anti-IHF-A5 (raised against SEQ ID NO: 352) was significant (FIG. 17).

It is to be understood that while the disclosure has been described inconjunction with the above embodiments, that the foregoing descriptionand examples are intended to illustrate and not limit the scope of thedisclosure. Other aspects, advantages and modifications within the scopeof the disclosure will be apparent to those skilled in the art to whichthe disclosure pertains.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. All nucleotide sequencesprovided herein are presented in the 5′ to 3′ direction.

The embodiments illustratively described herein may suitably bepracticed in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, theterms “comprising,” “including,” containing,” etc. shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the disclosure.

Thus, it should be understood that although the present disclosure hasbeen specifically disclosed by specific embodiments and optionalfeatures, modification, improvement and variation of the embodimentstherein herein disclosed may be resorted to by those skilled in the art,and that such modifications, improvements and variations are consideredto be within the scope of this disclosure. The materials, methods, andexamples provided here are representative of particular embodiments, areexemplary, and are not intended as limitations on the scope of thedisclosure.

The scoped of the disclosure has been described broadly and genericallyherein. Each of the narrower species and subgeneric groupings fallingwithin the generic disclosure also form part of the disclosure. Thisincludes the generic description with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatembodiments of the disclosure may also thereby be described in terms ofany individual member or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

What is claimed is:
 1. A method to prevent formation of or to dissolve abiofilm in a subject in need thereof, comprising administering to thesubject an isolated polypeptide consisting of the amino acid sequence ofRPGRNPKTGDVVPVSARRVV (SEQ ID NO: 352) or an equivalent thereof, whereinthe equivalent consists of the amino acid sequence of SEQ ID NO: 352 andeither or both of the following: (i) up to 20 random amino acids on theamine terminus of the amino acid sequence of SEQ ID NO: 352; or (ii) upto 15 random amino acids on the carboxy terminus of the amino acidsequence of SEQ ID NO: 352, and wherein the subject has chronicobstructive pulmonary disease (COPD), chronic cough, cystic fibrosis(CF), community acquired pneumonia (CAP), or a combination thereof. 2.The method of claim 1, comprising administering to the subject anisolated polypeptide consisting of the amino acid sequence of SEQ ID NO:352.
 3. The method of claim 1, wherein the subject is selected from thegroup of: humans, simians, rats, mice, chinchilla, canine, leporids,livestock, sport animals, or pets.
 4. The method of claim 1, wherein thebiofilm comprises a DNABII protein.
 5. The method of claim 4, whereinthe DNABII protein comprises a histone-like protein from E. coli strainU93 (HU) or an integration host factor (IHF) protein.
 6. The method ofclaim 1, wherein the biofilm is produced by a microorganism selectedfrom one or more of the group of: Haemophilus influenzae, Streptococcusmutans, Staphylococcus aureus, Moraxella catarrhalis, Streptococcuspneumonia, Pseudomonas aeruginosa, Neisseria gonorrhoeae, UropathogenicEscherichia coli, Staphylococcus epidermidis, Haemophilus influenzae(nontypeable)(NTHI), Streptococcus agalactiae, Neisseria meningitidis,Treponema denticola, Treponema pallidum, Burkholderia cepacia,Burkholderia pseudomallei, Streptococcus pyogenes, Mycobacteriumtuberculosis, Porphyromonas gingivalis, Aggregatibacteractinomycetemcomitans, Borrelia burgdorferi, Escherichia coli,Salmonella enterica serovar, Vibrio cholerae, Helicobacter pylori, orEnterococcus faecalis.
 7. The method of claim 1, further comprisingadministering an effective amount of one or more of the followingconcurrently or sequentially: an antibiotic, an antimicrobial, anantigenic peptide, an adjuvant, a DNase enzyme, or an antibody.
 8. Themethod of claim 1, wherein the administration is selected from the groupof: locally to the site of the infection, by direct injection, byinhalation, transdermally, urethrally, sublingually, rectally,vaginally, ocularly, subcutaneous, intramuscularly, intraperitoneally,intranasally, or orally.
 9. The method of claim 1, wherein the isolatedpolypeptide is administered in a composition further comprising apharmaceutically acceptable carrier, or wherein the isolated polypeptideis administered in a composition further comprising a solid phasecarrier selected from the group of: an implant, a stent, a paste, a gel,a dental implant, or a medical implant.
 10. The method of claim 1,wherein the isolated polypeptide is conjugated to a detectable agent.11. The method of claim 1, wherein the subject has been selected for themethod by assaying a sample isolated from the subject for infection by amicroorganism selected from one or more of the group of: Haemophilusinfluenzae, Streptococcus mutans, Staphylococcus aureus, Moraxellacatarrhalis, Streptococcus pneumonia, Pseudomonas aeruginosa, Neisseriagonorrhoeae, Uropathogenic Escherichia coli, Staphylococcus epidermidis,Haemophilus influenzae (nontypeable)(NTHI), Streptococcus agalactiae,Neisseria meningitidis, Treponema denticola, Treponema pallidum,Burkholderia cepacia, Burkholderia pseudomallei, Streptococcus pyogenes,Mycobacterium tuberculosis, Porphyromonas gingivalis, Aggregatibacteractinomycetemcomitans, Borrelia burgdorferi, Escherichia coli,Salmonella enterica serovar, Vibrio cholerae, Helicobacter pylori, orEnterococcus faecalis.
 12. The method of claim 1, further comprisingadministering to the subject an effective amount of an antibody orantigen binding fragment that specifically recognizes or binds thepolypeptide of SEQ ID NO:
 352. 13. The method of claim 2, wherein thebiofilm comprises a DNABII protein.
 14. The method of claim 13, whereinthe DNABII protein comprises a histone-like protein from E. coli strainU93 (HU) or an integration host factor (IHF) protein.
 15. The method ofclaim 2, wherein the biofilm is produced by a microorganism selectedfrom one or more of the group of: Haemophilus influenzae, Streptococcusmutans, Staphylococcus aureus, Moraxella catarrhalis, Streptococcuspneumonia, Pseudomonas aeruginosa, Neisseria gonorrhoeae, UropathogenicEscherichia coli, Staphylococcus epidermidis, Haemophilus influenzae(nontypeable)(NTHI), Streptococcus agalactiae, Neisseria meningitidis,Treponema denticola, Treponema pallidum, Burkholderia cepacia,Burkholderia pseudomallei, Streptococcus pyogenes, Mycobacteriumtuberculosis, Porphyromonas gingivalis, Aggregatibacteractinomycetemcomitans, Borrelia burgdorferi, Escherichia coli,Salmonella enterica serovar, Vibrio cholerae, Helicobacter pylori, orEnterococcus faecalis.
 16. The method of claim 2, wherein the isolatedpolypeptide is administered in a composition further comprising apharmaceutically acceptable carrier, or wherein the isolated polypeptideis administered in a composition further comprising a solid phasecarrier selected from the group of: an implant, a stent, a paste, a gel,a dental implant, or a medical implant.
 17. The method of claim 1,wherein the subject has chronic obstructive pulmonary disease (COPD).18. The method of claim 1, wherein the subject has chronic cough. 19.The method of claim 1, wherein the subject has cystic fibrosis (CF). 20.The method of claim 1, wherein the subject has a community acquiredpneumonia (CAP).
 21. The method of claim 2, wherein the subject haschronic obstructive pulmonary disease (COPD).
 22. The method of claim 2,wherein the subject has chronic cough.
 23. The method of claim 2,wherein the subject has cystic fibrosis (CF).
 24. The method of claim 2,wherein the subject has a community acquired pneumonia (CAP).